TWI403586B - Compositions and methods for identifying response targets and treating flavivirus infection responses - Google Patents

Abstract

Cellular receptors are identified that induce plasma leakage and other negative effects when infected with flaviviruses, such as dengue virus or Japanese encephamyelitis virus. Using fusion proteins disclosed herein, the receptors to which a pathogen, such as flavivirus, binds via glycan binding are determined. Once the receptors are determined, the effect of binding to a particular receptor may be determined, wherein targeting of the receptors causing a particular symptom may be targeted by agents that interrupt binding of the pathogen to the receptor. Accordingly, in the case of dengue virus and Japanese encephamyelitis virus, TNF-α is released when the pathogen binds to the DLVR1/CLEC5A receptor. Interrupting the DLVR1/CLEC5A receptor with monoclonal antibodies reduced TNF-α secretion without affecting secretion of cytokines responsible for viral clearance thereby increasing survival rates in infected mice from nil to around 50%.

Description

用於偵測目標反應與治療黃病毒感染症狀之化合物及方法Compound and method for detecting target response and treating symptoms of flavivirus infection

本申請案陳請美國專利申請案序號11/469,270號(2006年8月31日申請)，以及美國臨時專利申請案序號60/713,463號(2005年8月31日申請)之優先權以及權益，上述各申請案之揭示內容全文皆以參考資料方式納入本說明書中。Priority and benefit of U.S. Patent Application Serial No. 11/469,270 (filed on Aug. 31, 2006) and U.S. Provisional Patent Application Serial No. 60/713,463 (filed on Aug. 31, 2005). The entire disclosure of each of the above applications is incorporated herein by reference.

本發明係由台灣國家科學委員會94F008-5、NSC 95-2320-B-010010及NSC 95-3112-B-010-017之計畫所支持。本發明亦由台灣中央研究院94M002-1與國立陽明大學95A-CT8G02之計畫所支持。The present invention is supported by the National Science Council of Taiwan 94F008-5, NSC 95-2320-B-010010, and NSC 95-3112-B-010-017. The invention is also supported by the Taiwan Central Research Institute 94M002-1 and the National Yangming University 95A-CT8G02 program.

本發明係關於一種基於免疫受體(特別是DLVR1/CLEC5A)與病原之交互作用之各種應用。The present invention relates to various applications based on the interaction of immunoreceptors (especially DLVR1/CLEC5A) with pathogens.

在本說明書中所參照之任何先前技術皆不或不應被視做承認或以任何形式暗示該先前技術在任何國家構成通識之一部分。本文所引用之所有文獻，其全文皆以參考資料方式具體納入本說明書中。Any prior art referred to in this specification is not, or should be, construed as an admission that the prior art constitutes a part of the general knowledge in any country. All documents cited herein are hereby incorporated by reference in their entirety.

免疫系統能使宿主生物區辨本身及非本身抗原，以及識別及清除侵入之病原體。後天性免疫系統(adaptive immunity system)依存於高度多形(highly polymorphic)分子，諸如主要組織相容性複合體(MHC)之第I類及第II類抗原、T細胞受體及B細胞受體，以向T細胞及B細胞呈遞抗原，因而導致免疫系統之活化。先天免疫系統識別此等多樣性抗原之機制未明，直到Janeway提出模式識別受體(pattern recognition receptors，PRRs)之觀念(Janeway,1989,Cold Spring Harb Symp Quant Biol 54 Pt 1,1-13)。該假說後來藉由鑑定出由下列受體識別之病原體相關性分子模式(PAMPs)而證明為正確，該等受體為：類鐸受體(TOLL-like receptors)(Aderem and Ulevitch,2000 Nature 406,782-7；Akira and Takeda,2004,Nat Rev Immunol 4,499-511；Athman and Philpott,2004,Curr Opin Microbiol 7,25-32)、凝集素(lectin)受體(Cambi and Figdor,2003,Curr Opin Cell Biol 15,539-46)、類免疫球蛋白(類Ig)受體(Daws et al.,2003,J Immunol 171,594-9)、及NOD蛋白(Athman and Philpott,2004,Curr Opin Microbiol 7,25-32)、以及其他(Liu et al.,2001,J Biol Chem 276,34686-94；McDonald et al.,2005,J Biol Chem 280,20177-80)。The immune system enables the host biota to distinguish itself from non-self antigens, as well as to identify and remove invading pathogens. The adaptive immunity system is dependent on highly polymorphic molecules such as major histocompatibility complex (MHC) class I and class II antigens, T cell receptors and B cell receptors. To present antigen to T cells and B cells, thus leading to activation of the immune system. The mechanism by which the innate immune system recognizes these diverse antigens is unclear until Janeway proposes the concept of pattern recognition receptors (PRRs) (Janeway, 1989, Cold Spring Harb Symp Quant Biol 54 Pt 1, 1-13). This hypothesis was later proved to be correct by identifying pathogen-associated molecular patterns (PAMPs) recognized by the following receptors: TOLL-like receptors (Aderem and Ulevitch, 2000 Nature 406,782) -7; Akira and Takeda, 2004, Nat Rev Immunol 4, 499-511; Athman and Philpott, 2004, Curr Opin Microbiol 7, 25-32), lectin receptor (Cambi and Figdor, 2003, Curr Opin Cell Biol 15,539-46), immunoglobulin-like (Ig-like) receptors (Daws et al., 2003, J Immunol 171, 594-9), and NOD proteins (Athman and Philpott, 2004, Curr Opin Microbiol 7, 25-32), And others (Liu et al., 2001, J Biol Chem 276, 34686-94; McDonald et al., 2005, J Biol Chem 280, 20177-80).

除了由類鐸受體所識別且經充分鑑定特徵之PAMPs(Akira and Takeda,2004,Nat Rev Immunol 4,499-511)之外，新近之研究顯示宿主免疫系統可經由特異性碳水化合物抗原而識別侵入之病原體。舉例言之，甘露糖受體可識別在病原體表面表現之具高甘露糖分子部分(Stahl and Ezekowitz,1998,Curr Opin Immunol 10,50-5)，同時樹狀細胞凝集素-1(Dectin-1)受體可特異性地與β-葡聚糖結合，該β-葡聚糖為真菌壁表面之多醣體之主要骨架(Brown and Gordon,2001,Nature 413,36-7；Herre et al.,2004,Mol Immunol 40,869-76)。此等結果暗示與病原體相關之碳水化合物結構為由免疫細胞之先天性免疫受體所識別之標靶之一。In addition to PAMPs (Akira and Takeda, 2004, Nat Rev Immunol 4, 499-511), which are recognized by steroid-like receptors and are well characterized, recent studies have shown that the host immune system recognizes invasion through specific carbohydrate antigens. Pathogen. For example, the mannose receptor recognizes a high mannose moiety on the surface of a pathogen (Stahl and Ezekowitz, 1998, Curr Opin Immunol 10, 50-5), while dendritic cell lectin-1 (Dectin-1) The receptor specifically binds to β-glucan, which is the main skeleton of the polysaccharide on the surface of the fungal wall (Brown and Gordon, 2001, Nature 413, 36-7; Herre et al., 2004, Mol Immunol 40, 869-76). These results suggest that the carbohydrate structure associated with the pathogen is one of the targets recognized by the innate immune receptors of immune cells.

靈芝類(Ganoderma )及冬蟲夏草類(Cordyceps )真菌為在中國為最盛行服用之草藥。自靈芝(Ganoderma lucidum ，亦稱為Ling zhi、Reishi)萃取出之多醣體曾被用於傳統中藥而作為抗腫瘤劑及免疫調節劑(Lien,1990,Prog Drug Res 34,395-420；Wang et al.,2002,Bioorg Med Chem 10,1057-62；Shiao,2003,Chem Rec 3,172-80)，而從冬蟲夏草(Cordyceps sinensis )(Cordyceps、Caterpillar fungus)萃取出者則顯示會改變細胞凋亡自穩態(apoptotic homeostasis)，並會改善呼吸、腎臟、及心血管功能(Buenz et al.,2005,J Ethnopharmacol 96,19-29；Zhu et al.,1998,J Altern Complement Med 4,289-303；Zhu et al.,1998,J Altern Complement Med 4,429-57)，以及增加全身對於胰島素之敏感性(Balon et al.,2002,J Altern Complement Med 8,315-23)。不過，萃取物之多醣體組成於多醣體係萃取自不同來源、不同菌株、及於不同生長條件下培養時皆會發生變化。Ganoderma (Ganoderma) class and Cordyceps (Cordyceps) fungi in China for the taking of the most popular herbs. From Ganoderma lucidum (Ganoderma lucidum, also known as Ling zhi, Reishi) of extracted polysaccharides have been used in traditional Chinese medicine as an antitumor agent and an immunomodulator (Lien, 1990, Prog Drug Res 34,395-420; Wang et al. , 2002, Bioorg Med Chem 10, 1057-62; Shiao, 2003, Chem Rec 3, 172-80), while extracts from Cordyceps sinensis (Cordyceps, Caterpillar fungus) showed altered apoptotic homeostasis ( Apoptotic homeostasis) and will improve respiratory, renal, and cardiovascular functions (Buenz et al., 2005, J Ethnopharmacol 96, 19-29; Zhu et al., 1998, J Altern Complement Med 4, 289-303; Zhu et al. , 1998, J Altern Complement Med 4, 429-57), and increased systemic sensitivity to insulin (Balon et al., 2002, J Altern Complement Med 8, 315-23). However, the polysaccharide composition of the extract changes when the polysaccharide system is extracted from different sources, different strains, and cultured under different growth conditions.

仰賴高效液相層析(HPLC)及質子-核磁共振之分析方法曾被用於研究單離自靈芝(Ganoderma lucidum )及冬蟲夏草(Cordyceps sinensis )之多醣體之成分(He and Seleen,2004,Int.J.Med.Mushrooms 6,253)。不過，高效液相層析圖係基於與靈芝酸(ganoderic acid)A及C(靈芝之二種主要三萜化合物)或腺苷之比較。依據質譜仍難以知道萃取物是否含有多醣體之活性成分。Analytical methods relying on high performance liquid chromatography (HPLC) and proton-NMR have been used to study the composition of polysaccharides from Ganoderma lucidum and Cordyceps sinensis (He and Seleen, 2004, Int. J.Med.Mushrooms 6,253). However, HPLC is based on comparison with ganoderic acid A and C (the two major triterpenoids of Ganoderma lucidum) or adenosine. It is still difficult to know whether the extract contains the active ingredient of the polysaccharide according to the mass spectrum.

本發明辨識出在受到黃病毒(諸如，登革熱病毒(Dengue virus)或日本腦脊髓炎病毒(Japanese encephamyelitis virus))感染時會誘發細胞滲漏及其它負作用之細胞受體。使用本發明所揭露之融合蛋白，其判定出病原體(諸如，黃病毒)會經由糖苷鍵結而結合之受體。一旦判定出該等受體，即可判定結合特定受體之作用，其中對於造成特定症狀之受體的尋靶可由能夠阻斷病原體與受體結合之試劑靶向。因此，就登革熱病毒及日本腦脊髓炎病毒而言，TNF-α會在病原體結合DLVR1/CLEC5A受體時釋出。以單株抗體阻斷DLVR1/CLEC5A受體可減少TNF-α之分泌，且同時不會影響負責進行病毒清除之細胞激素的分泌，因而使經感染小鼠之存活率由零增加至約50%。The present invention recognizes cellular receptors that induce cell leakage and other negative effects when infected with a flavivirus such as Dengue virus or Japanese encephamyelitis virus. The fusion protein disclosed in the present invention is used to determine a receptor to which a pathogen (such as a flavivirus) binds via glycosidation. Once the receptors are determined, the effect of binding to a particular receptor can be determined, wherein targeting of the receptor that causes the particular condition can be targeted by an agent capable of blocking the binding of the pathogen to the receptor. Therefore, in the case of dengue virus and Japanese encephalomyelitis virus, TNF-α is released when the pathogen binds to the DLVR1/CLEC5A receptor. Blocking DLVR1/CLEC5A receptor by monoclonal antibody can reduce the secretion of TNF-α without affecting the secretion of cytokines responsible for viral clearance, thus increasing the survival rate of infected mice from zero to about 50%. .

根據本說明書揭露之特徵，本發明提供一種方法，其包含取得融合蛋白質族群(complement)(其中各融合蛋白質包含受體的結合區域(domain)以及提供受質(substrate)固定之區域)，使融合蛋白質與病原體接觸以確認該病原體是否可與該融合蛋白質族群中至少一融合蛋白質上之結合區域結合，及偵測該病原體是否與該融合蛋白質結合。「融合蛋白質族群」乙辭代表至少一受體的許多不同結合區域。In accordance with features disclosed herein, the present invention provides a method comprising obtaining a fusion protein population (wherein each fusion protein comprises a binding domain of a receptor and providing a substrate-fixed region), such that the fusion The protein is contacted with the pathogen to confirm whether the pathogen binds to a binding region on at least one of the fusion protein populations and whether the pathogen binds to the fusion protein. The "fusion protein group" is a number of different binding regions of at least one receptor.

根據本說明書揭露之特徵，本發明提供一種方法，其包含取得易受病原體感染之細胞，剔除至少一細胞受體基因，使該等細胞與該病原體接觸，及測量該等細胞之細胞激素分泌量。In accordance with features disclosed herein, the present invention provides a method comprising: obtaining a cell susceptible to infection by a pathogen, removing at least one cell receptor gene, contacting the cell with the pathogen, and measuring the amount of cytokine secretion by the cell. .

根據本說明書揭露之特徵，本發明提供一種方法，其包含鑑定至少一可結合病原體所展示之配體(ligand)的細胞受體，以及對經該病原體感染之動物投予試劑，以阻斷該配體與該受體之結合而調控該病原體之作用。In accordance with features disclosed herein, the present invention provides a method comprising identifying at least one cellular receptor that binds to a ligand exhibited by a pathogen, and administering an agent to the animal infected with the pathogen to block the The binding of the ligand to the receptor modulates the action of the pathogen.

根據本說明書揭露之特徵，本發明提供一種方法，其包含提供有效量的試劑，以調節感染動物之病原體的作用，以調節該病原體對該動物上的作用。該試劑係針對至少一該感染動物原有細胞之細胞受體，以防止該受體與該病原體所展示之配體結合。In accordance with features disclosed herein, the present invention provides a method comprising providing an effective amount of an agent to modulate the effects of a pathogen of an infected animal to modulate the effect of the pathogen on the animal. The reagent is directed to at least one cellular receptor of the original cell of the infected animal to prevent binding of the receptor to the ligand exhibited by the pathogen.

根據本說明書揭露之特徵，本發明提供一種方法，其包含提供有效量之抗DLVR1/CLEC5A抗體給予受登革熱病毒感染之動物，其中該抗DLVR1/CLEC5A抗體能防止由登革熱病毒顆粒所呈現之配體與DLVR1/CLEC5A受體結合，其中TNF-α之分泌可受到抑制。In accordance with features disclosed herein, the present invention provides a method comprising administering an effective amount of an anti-DLVR1/CLEC5A antibody to an animal infected with a dengue virus, wherein the anti-DLVR1/CLEC5A antibody prevents a ligand presented by the dengue virus particle Binding to the DLVR1/CLEC5A receptor, wherein secretion of TNF-α can be inhibited.

根據本說明書揭露之特徵，本發明提供一種方法，其包含提供有效量之抗DLVR1/CLEC5A抗體給予受日本腦脊髓炎病毒感染之動物，其中該抗DLVR1/CLEC5A抗體能防止由日本腦脊髓炎病毒所呈現之配體與DLVR1/CLEC5A受體結合，其中TNF-α之分泌可受到抑制。In accordance with features disclosed herein, the present invention provides a method comprising administering an effective amount of an anti-DLVR1/CLEC5A antibody to an animal infected with a Japanese encephalomyelitis virus, wherein the anti-DLVR1/CLEC5A antibody prevents the Japanese encephalomyelitis virus The ligand presented binds to the DLVR1/CLEC5A receptor, wherein secretion of TNF-[alpha] can be inhibited.

根據本說明書揭露之特徵，本發明提供一種方法，其包含提供有效量之試劑給予受登革熱病毒感染之動物，其中該試劑可至少部分抑制至少一種促發炎性細胞激素之分泌，且不影響干擾素-α(Interferon-α)之分泌。In accordance with features disclosed herein, the present invention provides a method comprising administering an effective amount of an agent to an animal infected with a dengue virus, wherein the agent at least partially inhibits secretion of at least one pro-inflammatory cytokine without affecting interferon -α (Interferon-α) secretion.

根據本說明書揭露之特徵，本發明提供一種小鼠，其包含易受登革熱病毒感染之小鼠，以及sh-RNA顆粒以剔除該小鼠體內之DLVR1/CLEC5A受體。In accordance with features disclosed herein, the present invention provides a mouse comprising a mouse susceptible to dengue virus infection, and sh-RNA particles to knock out the DLVR1/CLEC5A receptor in the mouse.

根據本說明書揭露之特徵，本發明提供一種組合物，其包含醫藥製備物，包含有效量之抗體，該抗體係抗動物體內至少一細胞受體，以調節該動物體內病原體感染之作用。該調節至少包含抑制該動物細胞之促發炎性細胞激素的分泌，且不影響可造成病毒清除之細胞激素的分泌。In accordance with features disclosed herein, the present invention provides a composition comprising a pharmaceutical preparation comprising an effective amount of an antibody against at least one cellular receptor in an animal to modulate pathogen infection in the animal. The modulation comprises at least inhibiting secretion of an inflammatory cytokine that inhibits the cells of the animal and does not affect secretion of cytokines that cause viral clearance.

根據本說明書揭露之特徵，本發明提供一種組合物，其包含醫藥製備物，包含有效量之抗體，該抗體係對抗受登革熱病毒感染之動物之DLVR1/CLEC5A受體，以調節該動物體內登革熱病毒感染之作用。該調節至少包含抑制該動物細胞之促發炎性細胞激素的分泌，且不影響可造成病毒清除之細胞激素的分泌。In accordance with features disclosed herein, the present invention provides a composition comprising a pharmaceutical preparation comprising an effective amount of an antibody against a DLVR1/CLEC5A receptor of a dengue virus-infected animal to modulate dengue virus in the animal The role of infection. The modulation comprises at least inhibiting secretion of an inflammatory cytokine that inhibits the cells of the animal and does not affect secretion of cytokines that cause viral clearance.

在下列本發明具體例之詳述中，其參照附呈之圖式閱讀時更為明瞭，其中類似之參照數目代表類似之元件，且其中係以說明方式顯示可實施本發明之特定具體例。此等具體例係以足夠之細節進行敘述，以使一般技藝人士可據以實施本發明，且需明瞭者，可使用其他具體例，且可在不偏離本發明範圍之情形下進行邏輯、機械、生物、電學、功能、及其它之改變。下文之詳述因此不應被視為任何限制，而本發明之範圍僅由附呈之申請專利範圍定義。在本文中，「或」乙辭應可被理解為定義一種論理上之選言，且不應被視為一種排他性選言，除非其被標示為「或不」(xor)乙辭。In the following detailed description of the embodiments of the invention, reference to the claims The specific examples are described in sufficient detail to enable those skilled in the art to practice the invention, and the invention may be practiced, and other specific embodiments may be utilized and may be practiced without departing from the scope of the invention. , biology, electricity, function, and other changes. The detailed description below is therefore not to be considered as limiting, and the scope of the invention is defined only by the scope of the appended claims. In this context, the word "or" should be understood as defining an arbitrarily chosen statement and should not be considered an exclusive choice unless it is marked as "xor".

在一具體例中，本發明提供一種融合蛋白質，其包含先天性免疫受體之碳水化合物識別區域及異源多肽。先天性免疫受體意指：1)由白血球受體複合物(LRC)中之基因及人類染色體19上之LRC-相關基因所編碼之受體，其包括，但不限於CD66家族(CEACAM1及PSG1)、SIGLEC家族、NGK7、FCGRT、ILT/LILRA/LILRB(CD85)家族、LAIR家族、KIR(CD158)家族(包括KIR2DL亞家族、KIR2DS亞家族及KIR3DL亞家族)、FCAR(CD89)、NKp46(NCR1)及GPVI(GP6)；以及In one embodiment, the invention provides a fusion protein comprising a carbohydrate recognition region of an innate immune receptor and a heterologous polypeptide. Innate immune receptor means: 1) a receptor encoded by a gene in the white blood cell receptor complex (LRC) and an LRC-related gene on human chromosome 19, including, but not limited to, the CD66 family (CEACAM1 and PSG1) ), SIGLEC family, NGK7, FCGRT, ILT/LILRA/LILRB (CD85) family, LAIR family, KIR (CD158) family (including KIR2DL subfamily, KIR2DS subfamily and KIR3DL subfamily), FCAR (CD89), NKp46 (NCR1) ) and GPVI (GP6);

2)由在人類染色體12上之天然殺手受體複合物(NKC)中之基因所編碼之受體，其包括，但非限於，MAFA-L(KLRG1)、A2M、NKR-P1A(KLRB1)、LLt1(CLEC2D)、CD69(CLEC2C)、KLRF1、AICL(CLEC2B)、CLEC-2(CLECFS2)、Lox-1(OLR1)、CD94(KLRD1)、NKG2-D(KLRK1)、NKG2-F(KLRC4)、NKG2-E(KLRC3)、NKG2-C(KLRC2)、NKG2A(KLRC1)、Ly49L(KLRA1)及PRB3；以及2) Receptors encoded by genes in the natural killer receptor complex (NKC) on human chromosome 12, including, but not limited to, MAFA-L (KLRG1), A2M, NKR-P1A (KLRB1), LLt1 (CLEC2D), CD69 (CLEC2C), KLRF1, AICL (CLEC2B), CLEC-2 (CLECFS2), Lox-1 (OLR1), CD94 (KLRD1), NKG2-D (KLRK1), NKG2-F (KLRC4), NKG2-E (KLRC3), NKG2-C (KLRC2), NKG2A (KLRC1), Ly49L (KLRA1) and PRB3;

3)所有人類及小鼠C-型凝集素(CLEC)家族基因、所有人類之類唾液酸結合性Ig(SIGLEC)基因、所有人類之在骨髓細胞上表現之激發受體(TREM)基因、所有人類之類TREM(TREML/TLT)基因、所有人類之類鐸受體(TLR)基因以及所有在人類染色體發現之人類之類Fc受體(包括FCRL1至FCLR6，亦包括FCLRM1及FCLRM2)基因。3) All human and mouse C-type lectin (CLEC) family genes, all human sialic acid-binding Ig (SIGLEC) genes, all human-excited receptors (TREM) genes expressed on bone marrow cells, all Genes such as the TREM (TREML/TLT) gene for humans, all human tick receptor (TLR) genes, and all human Fc receptors (including FCRL1 to FCLR6, including FCLRM1 and FCLRM2) found in human chromosomes.

使用人類基因組組織(Human Genome Organization，HUGO)搜索引擎網站可以查到能應用於本發明方法中之此等分類中的其他基因。亦可參考Immunological Reviews 2001 Vol.181：20-38中之基因座說明，該文全文以參考資料方式納入本文。Other genes that can be used in such classifications in the methods of the invention can be found using the Human Genome Organization (HUGO) search engine website. See also the description of the locus in Immunological Reviews 2001 Vol. 181:20-38, which is incorporated herein by reference in its entirety.

來自非人類物種之任何上述基因之直系同源物(orthologues)亦可用於本發明之方法中。Orthologues of any of the above genes from non-human species can also be used in the methods of the invention.

可被考量用於本發明之C-型凝集素基因包括，但不限於，下列人類基因：ASGR1、ASGR2(CLEC4H2)、CD207(CLEC4K/郎罕細胞特異性蛋白)、CD209(DC-SIGN/CLEC4L)、CD302(CLEC13A)、CLEC1A、CLEC1B(CLEC-2)、CLEC2A、CLEC2B、CD69、CLEC2D、CLEC2L、CLEC3A、CLEC3B、CLEC3O、CLEC3Q、CLEC4A、CLEC4C、CLEC4D(CLEC-6)、CLEC4E、CLEC4F(KCLR)、CLEC4G、CLEC4M(DC-SIGNR)、CD209、DLVR1/CLEC5A、CLEC6A(樹狀細胞凝集素-2)、CLEC7A(樹狀細胞凝集素-1)、CLEC9A、CLEC10A、CLEC11A、CLEC12A、CLEC14A、FCER2、KLRB1、KLRF1、LY75(DEC205)、MRC1、MRC1L1、MRC2(Endo180)、OLR1、PLA2R1、DCAL1及COLEC10。此等基因之任一者之同源物(homologues)亦在考量之列，來自其他種動物諸如小鼠(mice)及大鼠(rats)之直系同源物(orthologues)亦同。同源物(homologues)及直系同源物(orthologues)與列舉之C-型凝集素(lectin)基因之任一者之相同性可為50%、70%、80%、80.6%、83%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%或99.9%。被特別考量之直系同源物為小鼠中之庫佛氏(Kupffer)細胞受體(mKCR)基因(與人類CLEC4F同源)。C-type lectin genes that can be considered for use in the present invention include, but are not limited to, the following human genes: ASGR1, ASGR2 (CLEC4H2), CD207 (CLEC4K/Langham cell-specific protein), CD209 (DC-SIGN/CLEC4L) ), CD302 (CLEC13A), CLEC1A, CLEC1B (CLEC-2), CLEC2A, CLEC2B, CD69, CLEC2D, CLEC2L, CLEC3A, CLEC3B, CLEC3O, CLEC3Q, CLEC4A, CLEC4C, CLEC4D (CLEC-6), CLEC4E, CLEC4F (KCLR) ), CLEC4G, CLEC4M (DC-SIGNR), CD209, DLVR1/CLEC5A, CLEC6A (dendritic agglutinin-2), CLEC7A (dendritic agglutinin-1), CLEC9A, CLEC10A, CLEC11A, CLEC12A, CLEC14A, FCER2 , KLRB1, KLRF1, LY75 (DEC205), MRC1, MRC1L1, MRC2 (Endo180), OLR1, PLA2R1, DCAL1, and COLEC10. Homologues of any of these genes are also contemplated, as are orthologues from other species of animals such as mice and rats. The homology of homologues and orthologues to any of the listed C-type lectin genes may be 50%, 70%, 80%, 80.6%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% 99.7%, 99.8% or 99.9%. A particularly important ortholog is the Kupffer Cell Receptor (mKCR) gene in mice (homologous to human CLEC4F).

被考量用於本發明之TREM基因及TREML基因包括，但非限於，下列人類基因：類CD300抗原家族成員B(CD300LB)、類CD300抗原家族成員G(CD300LG)、TREM1、TREM2、TREML1(TLT1)、TREML2(TLT2)、TREML3(TLT3)及TREML4(TLT4)。此等基因之任一者之同源物亦在考量之列，來自其他種動物諸如小鼠及大鼠之直系同源物亦同。同源及直系同源物與此等列舉之TLR基因之任一者之相同性可為50%、70%、80%、80.6%、83%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%或99.9%。被特別考量之直系同源物包括來自小鼠之mTREM1、mTREM2、mTLT1及mTLT4。The TREM gene and TREML genes contemplated for use in the present invention include, but are not limited to, the following human genes: CD300-like antigen family member B (CD300LB), CD300-like antigen family member G (CD300LG), TREM1, TREM2, TREML1 (TLT1) , TREML2 (TLT2), TREML3 (TLT3), and TREML4 (TLT4). Homologues of any of these genes are also contemplated, as are orthologs from other species of animals such as mice and rats. The identity of homologous and orthologs to any of the listed TLR genes can be 50%, 70%, 80%, 80.6%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%. Orthologues that are specifically considered include mTREM1, mTREM2, mTLT1, and mTLT4 from mice.

被考量用於本發明之TLR基因包括，但不限於，下列人類基因：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR11、TLR12及TLR13。此等基因之任一者之同源物亦在考量之列，來自其他種動物諸如小鼠及大鼠之直系同源物亦同。同源物及直系同源物與所列舉之TLR基因之任一者之相同性可為50%、70%、80%、80.6%、83%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%或99.9%。The TLR genes contemplated for use in the present invention include, but are not limited to, the following human genes: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. Homologues of any of these genes are also contemplated, as are orthologs from other species of animals such as mice and rats. The identity of homologs and orthologs to any of the listed TLR genes can be 50%, 70%, 80%, 80.6%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

被考量用於本發明之SIGLEC基因包括，但不限於，下列人類基因：CD22、CD33、髓鞘相關醣蛋白(MAG)、SIGLEC5、SIGLEC6、SIGLEC7、SIGLEC8、SIGLEC9、SIGLEC10、SIGLEC11、SIGLEC12、SIGLEC13及唾液酸黏附素(SN)。此等基因之任一者之同源物亦在考量之列，來自其他種動物諸如小鼠及大鼠之直系同源物亦同。同源物及直系同源物與所列舉之SIGLEC基因之任一者之相同性可為50%、70%、80%、80.6%、83%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、99.1%、99.2%、99.3%、99.4%、99.5%、99.6%、99.7%、99.8%或99.9%。The SIGLEC genes contemplated for use in the present invention include, but are not limited to, the following human genes: CD22, CD33, myelin-associated glycoprotein (MAG), SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC13, and Sialic acid adhesin (SN). Homologues of any of these genes are also contemplated, as are orthologs from other species of animals such as mice and rats. The identity of homologs and orthologs to any of the listed SIGLEC genes can be 50%, 70%, 80%, 80.6%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

適用於本發明之其他先天性免疫受體包括在下述實施例中所記載者。Other innate immune receptors suitable for use in the present invention include those described in the Examples below.

融合蛋白質可包含先天性免疫受體之整個細胞外區域，該細胞外區域包括碳水化合物識別區域；或者融合蛋白質可包含該細胞外區域之一部分，包括碳水化合物識別區域；或者融合蛋白質可僅包含碳水化合物識別區域。The fusion protein may comprise an entire extracellular region of the innate immune receptor, the extracellular region comprising a carbohydrate recognition region; or the fusion protein may comprise a portion of the extracellular region, including a carbohydrate recognition region; or the fusion protein may comprise only carbohydrate water Compound identification area.

異源多肽可包含能與先天性免疫受體之碳水化合物識別區域融合，以致該異源多肽在活體內或試管中皆不會干擾碳水化合物區域與其同系(cognate)特異性碳水化合物之結合之任何多肽。異源多肽較佳為免疫球蛋白，諸如人類IgG1、IgG2a、IgG2b、IgG3、IgG4、IgM、IgE、IgD、IgAa及IgA2或者來自其他種動物之免疫球蛋白。較佳以免疫球蛋白之片段，例如IgG之Fc片段，做為異源多肽。在較佳之具體例中，該異源多肽為不會與人類Fc受體結合之免疫球蛋白變型。該等變型在本技術中已為熟知。舉例言之，可使用包含下列突變之人類IgG1 Fc變型：L234A、L235E、G237A及P331S。The heterologous polypeptide may comprise a fusion with a carbohydrate recognition region of an innate immune receptor such that the heterologous polypeptide does not interfere with the binding of the carbohydrate region to its cognate specific carbohydrate in vivo or in a test tube. Peptide. The heterologous polypeptide is preferably an immunoglobulin such as human IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgE, IgD, IgAa and IgA2 or immunoglobulins from other species of animal. Preferably, a fragment of an immunoglobulin, such as an Fc fragment of IgG, is used as a heterologous polypeptide. In a preferred embodiment, the heterologous polypeptide is an immunoglobulin variant that does not bind to a human Fc receptor. Such variations are well known in the art. For example, human IgG1 Fc variants comprising the following mutations: L234A, L235E, G237A and P331S can be used.

該異源多肽尚可包含一個或一個以上允許融合多肽固定在固體支撐物上或從複合物混合物中純化之功能區域。舉例言之，該異源多肽可包含His6 tag以允許融合蛋白質依照本技術中所熟知之方法附接在Ni-NTA固體支撐物上。再舉例言之，該異源多肽可包含麩胱甘肽-S-轉移酶(glutathione-S-transferase)區域，以致生成之融合蛋白質可吸附在，例如，麩胱甘肽珠粒或由麩胱甘肽衍生之微量滴定平皿中。The heterologous polypeptide may also comprise one or more functional regions that allow the fusion polypeptide to be immobilized on or purified from the solid support. For example, the heterologous polypeptide can comprise a His6 tag to allow the fusion protein to be attached to the Ni-NTA solid support in accordance with methods well known in the art. By way of further example, the heterologous polypeptide may comprise a glutathione-S-transferase region such that the resulting fusion protein is adsorbed, for example, by glutathione beads or by gluten Glycopeptide-derived microtiter plates.

該異源多肽亦可包含一分子或一分子以上之生物素或生物素衍生物。以此方式，融合蛋白質可固定在綴合有鏈黴抗生物素蛋白之固體支撐物上，或者可使綴合有鏈黴抗生物素蛋白之酶與融合蛋白質結合。The heterologous polypeptide may also comprise one or more molecules of biotin or biotin derivatives. In this way, the fusion protein can be immobilized on a solid support conjugated to streptavidin or the streptavidin-conjugated enzyme can be bound to the fusion protein.

融合蛋白質視需要在異源多肽與先天性免疫受體之碳水化合物識別區域之間復可包含連結子(linker)。該連結子可為肽類連結子，或者其可為非肽類連結子，諸如聚乙二醇。The fusion protein may optionally include a linker between the heterologous polypeptide and the carbohydrate recognition region of the innate immune receptor. The linker can be a peptide linker or it can be a non-peptide linker such as polyethylene glycol.

在融合蛋白質中，碳水化合物識別區域可為相對於異源多肽之C-端或者可為相對於異源多肽之N-端。In the fusion protein, the carbohydrate recognition region can be relative to the C-terminus of the heterologous polypeptide or can be relative to the N-terminus of the heterologous polypeptide.

本發明之融合蛋白質可藉由蛋白質製造技術中已知之任何方法製備。融合蛋白質較佳使用本技術熟知之重組DNA技術及蛋白質表現技術製備。舉例言之，編碼先天性免疫受體之碳水化合物識別區域之DNA，可藉由使用對該特定目標先天性免疫受體之碳水化合物識別區域具特異性之引子進行mRNA之反錄酶PCR(RT-PCR)而製備。然後可將生成之DNA，連同編碼異源多肽序列之DNA，以可譯讀之方式選殖入表現載體中。本發明所使用之表現載體典型地含有複製源、位於5’端(即其上游)之啟動子、並接續編碼融合蛋白質之DNA序列、轉錄終止序列及剩餘之載體。該等表現載體亦可包括本技術已知之其他DNA序列，例如提供表現產物安定性之安定性前導序列、提供表現產物之分泌之分泌前導序列以及允許調節或誘發融合蛋白質表現之序列。表現載體亦可含有允許使用病毒表現系統，諸如本技術熟知之桿狀病毒(baculovirus)表現系統，來表現融合蛋白質之病毒序列。可將表現載體引進宿主細胞，諸如微生物細胞、酵母細胞、哺乳動物細胞或昆蟲細胞。可將表現載體以裸DNA之形式引進細胞中，或者其可被包封在病毒(諸如桿狀病毒)中。可將表現載體保持在宿主細胞內，或者將表現載體整合入宿主細胞基因組中。The fusion proteins of the invention can be prepared by any method known in the art of protein production. The fusion protein is preferably prepared using recombinant DNA techniques and protein expression techniques well known in the art. For example, DNA encoding a carbohydrate recognition region of an innate immune receptor can be subjected to mRNA reverse transcription PCR (RT) by using a primer specific for the carbohydrate recognition region of the specific target innate immune receptor. Prepared by -PCR). The resulting DNA, along with the DNA encoding the heterologous polypeptide sequence, can then be ligated into the expression vector in a translatable manner. The expression vector used in the present invention typically contains a replication source, a promoter located at the 5' end (i.e., upstream thereof), and a DNA sequence encoding the fusion protein, a transcription termination sequence, and the remaining vector. Such expression vectors can also include other DNA sequences known in the art, such as a stability leader sequence that provides for product stability, a secretion leader sequence that provides secretion of the expression product, and sequences that allow for modulation or induction of expression of the fusion protein. The expression vector may also contain viral sequences that permit the use of viral expression systems, such as the baculovirus expression systems well known in the art, to express the fusion protein. The expression vector can be introduced into a host cell, such as a microbial cell, a yeast cell, a mammalian cell, or an insect cell. The expression vector can be introduced into the cell in the form of naked DNA, or it can be encapsulated in a virus such as a baculovirus. The expression vector can be maintained in the host cell or the expression vector can be integrated into the host cell genome.

表現載體較佳包含能使分泌前導序列加至融合蛋白質上之DNA序列，藉此使得融合蛋白質分泌至環繞宿主細胞之培養基中。然後可用本技術已知之技術將融合蛋白質從培養基中純化。舉例言之，若融合蛋白質包含IgG以做為異源多肽，則蛋白質A管柱可用於結合融合蛋白質，以允許融合蛋白質與周圍培養基中之其他蛋白質分離。The expression vector preferably comprises a DNA sequence which enables secretion of the leader sequence to the fusion protein, whereby the fusion protein is secreted into the culture medium surrounding the host cell. The fusion protein can then be purified from the culture medium using techniques known in the art. For example, if the fusion protein comprises IgG as a heterologous polypeptide, the Protein A column can be used to bind the fusion protein to allow separation of the fusion protein from other proteins in the surrounding medium.

融合蛋白質亦可藉由使用試管內表現系統諸如爪蟾卵母細胞表現系統，在試管中轉譯編碼融合蛋白質之mRNA而製造。The fusion protein can also be produced by translating the mRNA encoding the fusion protein in a test tube using an in vitro expression system such as the Xenopus oocyte expression system.

在一具體例中，融合蛋白質係分開製造，然後使用本技術已知之化學技術將該等偶合在一起。舉例言之，可將碳水化合物識別區域及異源多肽分開製造，然後藉由使用戊二醛而將彼此偶合。In one embodiment, the fusion proteins are made separately and then coupled together using chemical techniques known in the art. For example, the carbohydrate recognition region and the heterologous polypeptide can be separately produced and then coupled to each other by using glutaraldehyde.

製造融合蛋白質之後，融合蛋白質可用可檢測之標記諸如螢光物質、放射線標記、酶、酶受質、染劑、化學發光劑、磁珠、量子點或其他任何能直接或間接產生可檢測信號之部分予以標幟。本技術已知許多使此等可檢測標記與蛋白質綴合之方法。舉例言之，可將經N-羥基琥珀醯亞胺活化之染料，最佳為經N-羥基琥珀醯亞胺活化之螢光物質，藉由與融合蛋白質上之一級胺反應而與融合蛋白質綴合。After the fusion protein is produced, the fusion protein can be detected by a detectable label such as a fluorescent substance, a radiation label, an enzyme, an enzyme substrate, a dye, a chemiluminescent agent, a magnetic bead, a quantum dot or any other which can directly or indirectly generate a detectable signal. Partially marked. A number of methods are known in the art for conjugating such detectable labels to proteins. For example, the dye activated by N-hydroxysuccinimide may be preferably a fluorescent substance activated by N-hydroxysuccinimide, which is fused with a fusion protein by reacting with a primary amine on the fusion protein. Hehe.

在一些具體例中，使用本技術已知之方法將融合蛋白質生物素化，以致該融合蛋白質包含一個或多個生物素分子，或者一個或多個生物素衍生物分子。藉此方式，該融合蛋白質可附接於鏈黴抗生物素蛋白可檢測部分綴合物，諸如酶-鏈黴抗生物素蛋白綴合物。In some embodiments, the fusion protein is biotinylated using methods known in the art such that the fusion protein comprises one or more biotin molecules, or one or more biotin derivative molecules. In this way, the fusion protein can be attached to a streptavidin detectable moiety conjugate, such as an enzyme-streptavidin conjugate.

在一系列之具體例中，本發明之融合蛋白質可被用於測定在包含多醣體之組合物中是否存在特異性碳水化合物成分。該方法涉及使多醣體與能和多醣體之特異性碳水化合物成分結合之融合蛋白質接觸，然後測定融合蛋白質是否與組合物中之多醣體結合。舉例言之，已知CLEC7A(亦稱為樹狀細胞凝集素-1)之碳水化合物識別區域可與β-1,3-D-葡聚糖交互作用(參見Brown,G.D.and Gordon,S.,2001,Nature 413,36-7，該文獻全文以參考資料方式納入本文)。所以包含CLEC7A之碳水化合物識別區域之融合蛋白質結合至多醣體組合物表示該多醣體組合物包含β-1,3-D-葡聚糖。同樣地，既然囓齒動物之庫佛氏(Kupffer)細胞受體(KCR；與人類CLEC4F同源)對於D-半乳糖及N-乙醯基半乳糖胺具有高親和性，並能從血清中清除以D-半乳糖及D-岩藻糖為終端之醣蛋白(參見Fadden,A.J.,Holt,O.J.and Drickamer,K.(2003)；Glycobiology 13,529-37,該文獻全文以參考資料方式納入本文)，包含KCR之碳水化合物識別區域之融合蛋白質結合至多醣體組合物表示該多醣體組合物包含D-半乳糖或N-乙醯基半乳糖胺或以D-半乳糖為終端之醣蛋白或以D-岩藻糖為終端之醣蛋白。此外，CD209(亦稱為DC-SIGN及CLEC4L)以及CLEC4M(亦稱為DC-SIGNR及L-SIGN)皆可結合至Man₉ GlcNAc₂ Asn醣肽，但只有CD209可結合至具有終端岩藻糖殘基之聚糖，而CLEC4M則否(參見Guo et al(2004)Nat Struct Mol Biol 11、591-8)；所以CD209及CLEC4M之融合蛋白質可區分包含此等碳水化合物成分之多醣體組合物。所以本發明之方法及試劑可用於鑑定多醣體組合物之碳水化合物成分以及此等碳水化合物成分之相對量，例如，以「指紋辨識(fingerprint)」多醣體組合物之獨特細微特徵。舉例言之，本發明之方法及試劑可用於測定具有免疫調節活性之多醣體組合物之碳水化合物成分。In a series of specific examples, the fusion protein of the invention can be used to determine the presence or absence of a specific carbohydrate component in a composition comprising a polysaccharide. The method involves contacting the polysaccharide with a fusion protein capable of binding to a specific carbohydrate component of the polysaccharide, and then determining whether the fusion protein binds to the polysaccharide in the composition. For example, the carbohydrate recognition region of CLEC7A (also known as dendritic lectin-1) is known to interact with β-1,3-D-glucan (see Brown, GD and Gordon, S., 2001). , Nature 413, 36-7, which is incorporated herein in its entirety by reference. Therefore, binding of the fusion protein comprising the carbohydrate recognition region of CLEC7A to the polysaccharide composition means that the polysaccharide composition comprises β-1,3-D-glucan. Similarly, since the rodent Kupffer cell receptor (KCR; homologous to human CLEC4F) has high affinity for D-galactose and N-ethylmercaptogalactosamine, it can be cleared from serum. Glycoproteins terminated by D-galactose and D-fucose (see Fadden, AJ, Holt, OJ and Drickamer, K. (2003); Glycobiology 13, 529-37, which is incorporated herein by reference in its entirety) The binding of the fusion protein of the carbohydrate recognition region of KCR to the polysaccharide composition means that the polysaccharide composition comprises D-galactose or N-ethylmercaptogalactosamine or a glycoprotein terminated by D-galactose or D- Fucose is the terminal glycoprotein. In addition, CD209 (also known as DC-SIGN and CLEC4L) and CLEC4M (also known as DC-SIGNR and L-SIGN) can bind to Man ₉ GlcNAc ₂ Asn glycopeptide, but only CD209 can bind to terminal fucose The glycan of the residue, and CLEC4M (see Guo et al (2004) Nat Struct Mol Biol 11, 591-8); therefore, the fusion protein of CD209 and CLEC4M can distinguish the polysaccharide composition containing these carbohydrate components. Thus, the methods and reagents of the present invention can be used to identify the carbohydrate component of a polysaccharide composition and the relative amounts of such carbohydrate components, for example, to "fingerprint" the unique subtle characteristics of the polysaccharide composition. For example, the methods and reagents of the present invention can be used to determine the carbohydrate component of a polysaccharide composition having immunomodulatory activity.

此外，若已知表現融合蛋白質之碳水化合物識別區域所來自之先天性免疫受體之細胞為何種類別，則本發明之檢定分析(assay)將能顯示在身體內與所探討之多醣體結合之細胞之類別。該知識，例如，有助於揭露特定多醣體組合物(諸如單離自靈芝之多醣體)對於與該多醣體接觸之生物施予有利或有害作用之機制。在該具體例中，非必須知道該碳水化合物識別區域所結合之碳水化合物成分之類別。Furthermore, if it is known what type of cells expressing the innate immune receptor from which the carbohydrate recognition region of the fusion protein is derived, the assay of the present invention will be able to display in vivo binding to the polysaccharide in question. The type of cell. This knowledge, for example, helps to uncover the mechanism by which a particular polysaccharide composition, such as a polysaccharide isolated from Ganoderma lucidum, exerts a beneficial or deleterious effect on the organism in contact with the polysaccharide. In this specific example, it is not necessary to know the category of the carbohydrate component to which the carbohydrate recognition region is bound.

本發明之融合蛋白質與其同系(cognate)碳水化合物成分之結合，可藉由將包含多醣體之組合物固定於固體支撐物，然後使該固體支撐物與融合蛋白質接觸而進行。融合蛋白質之結合可藉由檢測在固體支撐物之表面是否存在融合蛋白質，例如，藉由檢測固體支撐物之表面是否存在異源多肽或檢測固體支撐物之表面是否存在碳水化合物識別區域而檢測。例如，若異源多肽與螢光物質綴合，則沖洗後固體支撐物之表面存在螢光物質表示融合蛋白質存在，其最終表示存在包含由融合蛋白質之碳水化合物識別區域所識別之特異性碳水化合物成分之多醣體。The binding of the fusion protein of the present invention to its cognate carbohydrate component can be carried out by immobilizing the composition comprising the polysaccharide onto a solid support and then contacting the solid support with the fusion protein. The binding of the fusion protein can be detected by detecting the presence or absence of a fusion protein on the surface of the solid support, for example, by detecting the presence or absence of a heterologous polypeptide on the surface of the solid support or detecting the presence or absence of a carbohydrate recognition region on the surface of the solid support. For example, if a heterologous polypeptide is conjugated to a fluorescent material, the presence of a fluorescent material on the surface of the solid support after rinsing indicates the presence of the fusion protein, which ultimately indicates the presence of a specific carbohydrate comprising the carbohydrate recognition region of the fusion protein. The polysaccharide of the ingredient.

在本文中，「固體支撐物」被界定為分子可經由共價鍵或非共價鍵附接之任何表面。該固體支撐物包括，但不限於，膜(例如，聚偏二氟乙烯(PVDF)膜)、塑膠(例如微量滴定平皿)、順磁珠(paramagnetic beads)、帶電紙、尼龍、Langmuir-Bodgett薄膜、官能化玻璃、鍺、矽、PTFE、聚苯乙烯、砷化鎵、金及銀。在本技術中任何已知之在表面能納入胺基、羧基、硫醇基或羥基之其他材料亦可列入考量。此包括具有任何拓樸結構之表面，該拓樸結構包括，但不限於，球表面、有溝槽表面及圓柱形表面，例如管柱。As used herein, a "solid support" is defined as any surface to which a molecule can be attached via a covalent bond or a non-covalent bond. The solid support includes, but is not limited to, a membrane (eg, a polyvinylidene fluoride (PVDF) membrane), a plastic (eg, a microtiter plate), paramagnetic beads, a charged paper, a nylon, a Langmuir-Bodgett film. , functionalized glass, ruthenium, osmium, PTFE, polystyrene, gallium arsenide, gold and silver. Any other material known in the art to incorporate an amine group, a carboxyl group, a thiol group or a hydroxyl group on the surface may also be considered. This includes surfaces having any topographical structure including, but not limited to, spherical surfaces, grooved surfaces, and cylindrical surfaces, such as tubular strings.

包含多醣體之組合物(在本文中亦稱為多醣體組合物)可非限定地為包括多醣體之任何組合物，該多醣體包括，例如，醣蛋白(包括蛋白多醣(proteoglycan))、醣脂質、肽醣(peptidoglycan)、微生物細胞壁、病毒粒子及真菌細胞壁。在其他具體例中，包含多醣體之組合物為在溶液中之游離多醣體，例如未附接於蛋白質或脂質之多醣體。在本文中，「多醣體」意指包含2個或2個以上單醣之碳水化合物分子。The composition comprising a polysaccharide (also referred to herein as a polysaccharide composition) may be, without limitation, any composition comprising a polysaccharide comprising, for example, a glycoprotein (including proteoglycan), sugar Lipids, peptidoglycan, microbial cell walls, virions, and fungal cell walls. In other specific examples, the composition comprising the polysaccharide is a free polysaccharide in solution, such as a polysaccharide that is not attached to a protein or lipid. As used herein, "polysaccharide" means a carbohydrate molecule comprising two or more monosaccharides.

包含多醣體之組合物在固體支撐物上之固定化例如可藉由將組合物中之多醣體生物素化，然後將其固定在綴合有鏈黴抗生物素蛋白之固體支撐物上而達成。此外，可將多醣體固定在，例如，經甲醇活化之PVDF膜上。尤被列入考量者為以「轉漬」之方式進行本發明之方法，其中使用固定在PVDF膜上之多醣體點。Immobilization of a composition comprising a polysaccharide onto a solid support can be achieved, for example, by biotinylating the polysaccharide in the composition and then immobilizing it on a solid support conjugated to streptavidin. . Further, the polysaccharide body can be immobilized, for example, on a methanol-activated PVDF membrane. In particular, it is considered that the method of the present invention is carried out in a "transfer" manner in which a polysaccharide spot fixed on a PVDF membrane is used.

在一些具體例中，融合蛋白質與固定之多醣體之結合可藉由下法檢測：使第二試劑(secondary reagent)結合至融合蛋白質，較佳結合至異源多肽，繼而檢測第二試劑之存在。舉例言之，可將生物素化融合蛋白質附接於綴合有鏈黴抗生物素蛋白之酶，並藉由加入能得到可檢測產物之受質來檢測酶之存在。未生物素化之融合蛋白質可用例如可結合至異源多肽之抗體(若異源多肽為IgG或IgG Fc，則抗體為諸如抗-IgG抗體)而檢測，其中二次抗體與酶綴合。舉例言之，若酶為辣根過氧化酶(HRP)，則融合蛋白質結合之檢測可用本技術已知之加強化學發光(ECL)技術進行。可將第二試劑復綴合至或改為綴合至可檢測標記，諸如螢光物質或放射性核。在本技術中尚知道許多可用於檢測所揭示之融合蛋白質與固體支撐物之結合之其他技術。In some embodiments, the binding of the fusion protein to the immobilized polysaccharide can be detected by binding a secondary reagent to the fusion protein, preferably to the heterologous polypeptide, and then detecting the presence of the second reagent. . For example, a biotinylated fusion protein can be attached to an enzyme conjugated to streptavidin, and the presence of the enzyme can be detected by the addition of a substrate capable of obtaining a detectable product. The unbiotinylated fusion protein can be detected, for example, by an antibody that binds to the heterologous polypeptide (if the heterologous polypeptide is IgG or IgG Fc, the antibody is, for example, an anti-IgG antibody), wherein the secondary antibody is conjugated to the enzyme. For example, if the enzyme is horseradish peroxidase (HRP), detection of fusion protein binding can be performed using enhanced chemiluminescence (ECL) techniques known in the art. The second agent can be complexed or conjugated to a detectable label, such as a fluorescent material or a radionuclide. A number of other techniques are known in the art for detecting the binding of the disclosed fusion proteins to solid supports.

尤被考量者為上述檢定分析可以多工化陣列模式進行。例如固體支撐物可被分隔成複數個空間上分離之位址，在該等位址上可結合有多個不同的組合物。然後將該固體支撐物與融合蛋白質接觸並檢測該融合蛋白質之結合。以此方式可以測定被固定之多醣體組合物中何者(若有)包含可與融合蛋白質之碳水化合物識別區域結合之特異性碳水化合物成分。In particular, the above-mentioned verification analysis can be performed in a multiplexed array mode. For example, a solid support can be separated into a plurality of spatially separated addresses at which a plurality of different compositions can be combined. The solid support is then contacted with the fusion protein and the binding of the fusion protein is detected. In this manner, it can be determined which of the immobilized polysaccharide compositions, if any, contains a specific carbohydrate component that binds to the carbohydrate recognition region of the fusion protein.

在另一具體例中，單一組合物被固定在固體支撐物上，該固體支撐物被分隔成複數個在空間上分離之位址。各位址然後與不同的融合蛋白質接觸，各不同的融合蛋白質包含不同的碳水化合物識別區域。沖洗以移除非特異性結合之材料後，然後可如上述檢測融合蛋白質之結合：檢測到之各結合反應之空間位址揭露所結合之融合蛋白質之類別。以此方式，可同時使用許多不同的融合蛋白質偵測組合物。在該具體例中，各融合蛋白質可包含相同的異源多肽，藉此允許使用單一第二試劑同時檢測在各位址之結合。例如，若各融合蛋白質包含IgG Fc以做為異源多肽，則可使用抗-IgG抗體或蛋白質A或蛋白質G檢測融合蛋白質之結合。In another embodiment, a single composition is immobilized on a solid support that is divided into a plurality of spatially separated sites. The sites are then contacted with different fusion proteins, each of which contains a different carbohydrate recognition region. After rinsing to remove non-specifically bound material, the binding of the fusion protein can then be detected as described above: the spatial address of each of the binding reactions detected reveals the type of fusion protein bound. In this way, many different fusion protein detection compositions can be used simultaneously. In this particular example, each fusion protein can comprise the same heterologous polypeptide, thereby allowing simultaneous detection of binding at a single site using a single second reagent. For example, if each fusion protein comprises an IgG Fc as a heterologous polypeptide, the binding of the fusion protein can be detected using an anti-IgG antibody or Protein A or Protein G.

本發明之融合蛋白質及方法可用於「指紋辨識(fingerprint)」任何包含多醣體之組合物之獨特細微特徵，該等組合物包括，但不限於得自草藥製劑之多醣體組合物，諸如單離自真菌：靈芝(Ganoderma lucidim)、冬蟲夏草(Cordyceps sinensis)及蘑菇(Lentinus edodes)以及得自植物霍山石斛(Dendrobium huoshanense)之含多醣體部分。特定而言，尤被考量者為使用本文所述之方法測定靈芝多醣體之F3多醣體部分之碳水化合物成分(參見Wang,et al(2002)Bioorg Med Chem 10,1057-62；Chen,et al(2004)Bioorg Med Chem 12,5595-601；Chien,et al(2004)Bioorg Med Chem 12,5603-9.；以及Hsu et al(2004)J Immunol 173,5989-99，各文獻全文以參考資料方式納入本文)。The fusion proteins and methods of the present invention can be used to "fingerprint" the unique subtle features of any polysaccharide-containing composition, including but not limited to polysaccharide compositions derived from herbal preparations, such as isolated From fungi: Ganoderma lucidim, Cordyceps sinensis and mushroom (Lentinus edodes) and the polysaccharide-containing fraction derived from the plant Dendrobium huoshanense. In particular, it is specifically contemplated to determine the carbohydrate component of the F3 polysaccharide portion of the Ganoderma lucidum polysaccharide using the methods described herein (see Wang, et al (2002) Bioorg Med Chem 10, 1057-62; Chen, et al (2004) Bioorg Med Chem 12, 5595-601; Chien, et al (2004) Bioorg Med Chem 12, 5603-9.; and Hsu et al (2004) J Immunol 173, 5989-99, the entire contents of which are incorporated by reference. Ways to incorporate this article).

本文提供之方法可被用於指紋辨識複合物混合物之獨特細微特徵，該複合物混合物包括許多不同的多醣體組合物，或者可用於只含單一多醣體種類，例如單一醣蛋白或單一多醣體之製劑。The methods provided herein can be used to fingerprint unique complex features of a mixture of complexes comprising a plurality of different polysaccharide compositions, or can be used to contain only a single polysaccharide species, such as a single glycoprotein or a single polysaccharide. preparation.

若已知表現碳水化合物識別區域所來自之先天性免疫受體之細胞之類別，則上述檢定分析將可揭露當將多醣體組合物引進體內時，體內會與多醣體結合之細胞。然後亦可能得到調節所鑑定之先天性免疫受體之活性之藥劑。例如，若先天性免疫受體與多醣體之交互作用在體內造成有益之作用，則可以製造擬似該多醣體之結構或加強該多醣體與該先天性免疫受體間之交互作用之藥劑。參見下文以「調節劑」為標題之段落。If a class of cells expressing an innate immune receptor from which the carbohydrate recognition region is derived is known, the above assay will reveal cells that bind to the polysaccharide when the polysaccharide composition is introduced into the body. It is then also possible to obtain an agent that modulates the activity of the identified innate immune receptor. For example, if the interaction between the innate immune receptor and the polysaccharide causes a beneficial effect in the body, an agent that mimics the structure of the polysaccharide or enhances the interaction between the polysaccharide and the innate immune receptor can be produced. See the paragraph titled "Regulators" below.

在另一系列具體例中，本發明之方法及融合蛋白質被用於測定在病原體表面展示之多醣體之類別(identity)，該等病原體諸如真菌細胞、細菌細胞或病毒(諸如具套膜病毒；且病毒亦非限定性地包括來自黃熱病毒科之病毒)。適用於本發明方法之黃熱病毒科病毒非限定性地包括黃熱病毒屬成員(諸如登革熱病毒(DV)、西尼羅熱病毒(WNV)、日本腦脊髓炎病毒(JEV)、黃熱病毒(YFV)及蜱傳腦脊髓炎病毒)或肝炎病毒屬成員(諸如C型肝炎病毒)。在一該具體例中，將融合蛋白質固定在固體支撐物上(例如若異源多肽為IgG或其片段，則使用由蛋白質A所衍生之固體支撐物)，然後使固體支撐物與包含所探究病原體之組合物接觸。沖洗後，使用例如第二試劑檢測病原體之結合，該第二試劑可以不會與融合蛋白質之結合競爭之方式，與病原體特異性地結合。舉例言之，可使用對病原體具特異性之二次抗體。然後第二試劑之結合可如上述檢測(例如使用綴合有HRP之二次抗體)或者使用與第二試劑結合之第三試劑進行檢測(舉例言之，若第二試劑為抗-病原體IgG，則使用與HRP綴合之抗-IgG抗體)。若檢測到二次試劑之結合，其顯示該病原體包含多醣體且該多醣體包含融合蛋白質之碳水化合物識別區域所識別之特異性碳水化合物成分。In another series of specific embodiments, the methods and fusion proteins of the invention are used to determine the identity of a polysaccharide displayed on the surface of a pathogen, such as a fungal cell, a bacterial cell, or a virus (such as a cannula virus; And the virus also includes, without limitation, a virus from the yellow fever virus family. Yellow fever virus viruses suitable for use in the methods of the invention include, without limitation, members of the yellow fever virus (such as dengue virus (DV), West Nile fever virus (WNV), Japanese encephalomyelitis virus (JEV), yellow fever virus). (YFV) and sputum encephalomyelitis virus) or members of the hepatitis virus (such as hepatitis C virus). In this embodiment, the fusion protein is immobilized on a solid support (for example, if the heterologous polypeptide is IgG or a fragment thereof, a solid support derived from protein A is used), and then the solid support and inclusion are explored. The composition of the pathogen is in contact. After rinsing, the binding of the pathogen is detected using, for example, a second reagent that specifically binds to the pathogen in a manner that does not compete with the binding of the fusion protein. For example, a secondary antibody specific for a pathogen can be used. The binding of the second reagent can then be detected as described above (eg, using a secondary antibody conjugated to HRP) or using a third reagent in combination with a second reagent (for example, if the second reagent is an anti-pathogen IgG, An anti-IgG antibody conjugated to HRP is then used. If a binding of the secondary agent is detected, it is shown that the pathogen comprises a polysaccharide and the polysaccharide comprises a specific carbohydrate component recognized by the carbohydrate recognition region of the fusion protein.

或者，該檢定分析可藉由將與病原體特異性結合之試劑固定在固體支撐物上而進行。例如將可與病原體結合之抗體固定在固體支撐物上，繼而與包含病原體之組合物接觸。然後使固體支撐物與融合蛋白質接觸，並如上述檢測融合蛋白質之結合(該融合蛋白質較佳不會和病原體競爭與固定試劑之結合)。舉例言之，若融合蛋白質之異源多肽為IgG Fc，則抗-IgG抗體可檢測融合蛋白質與病原體之結合；或者若融合蛋白質與可檢測之標記綴合，則該標記之檢測被用於檢測結合。Alternatively, the assay can be performed by immobilizing an agent that specifically binds to the pathogen on a solid support. For example, an antibody that binds to a pathogen is immobilized on a solid support and then contacted with a composition comprising the pathogen. The solid support is then contacted with the fusion protein and the binding of the fusion protein is detected as described above (the fusion protein preferably does not compete with the pathogen for binding to the immobilizing agent). For example, if the heterologous polypeptide of the fusion protein is IgG Fc, the anti-IgG antibody can detect binding of the fusion protein to the pathogen; or if the fusion protein is conjugated to a detectable label, the detection of the label is used for detection Combine.

被經常列入考量者為上述病原體檢定分析，例如同時使用複數種不同的融合蛋白質以多重方式進行。舉例言之，將可與病原體結合之抗體固定在固體支撐物上之複數個分開的位址；然後使固體支撐物與包含病原體之組合物接觸；接著使各特異性位址與不同的融合蛋白質接觸，各不同的融合蛋白質包含不同的碳水化合物識別區域。若各融合蛋白質包含相同的異源多肽，則融合蛋白質之結合可用能與異源多肽結合之單一試劑檢測。舉例言之，若異源多肽為IgG Fc，則抗-IgG抗體可被用於檢測融合蛋白質之結合。各結合反應之空間位址即可揭露出融合蛋白質之種類。或者，可使用固定在固體支撐物之空間分離位址上之複數種不同融合蛋白質進行多重檢定分析，其中使固體支撐物與包含病原體之組合物接觸，繼而使固體支撐物與能和病原體特異性結合之第二試劑接觸。舉例言之，若病原體為登革熱病毒，則第二試劑可為對抗E套膜蛋白質之抗體。如在上述所有檢定分析中者，可進行沖洗以從固體支撐物移除非特異性結合之物質。The pathogen assay described above is often considered, for example, using multiple different fusion proteins simultaneously in multiple ways. For example, an antibody that binds to a pathogen is immobilized on a plurality of separate sites on a solid support; the solid support is then contacted with a composition comprising the pathogen; then each specific site is mapped to a different fusion protein In contact, the different fusion proteins contain different carbohydrate recognition regions. If each fusion protein comprises the same heterologous polypeptide, the binding of the fusion protein can be detected using a single reagent that binds to the heterologous polypeptide. For example, if the heterologous polypeptide is an IgG Fc, an anti-IgG antibody can be used to detect binding of the fusion protein. The spatial address of each binding reaction reveals the type of fusion protein. Alternatively, multiple assays can be performed using a plurality of different fusion proteins immobilized on the spatial separation site of the solid support, wherein the solid support is contacted with a composition comprising the pathogen, which in turn allows the solid support to be specific to the pathogen The second reagent is combined with the contact. For example, if the pathogen is a dengue virus, the second agent can be an antibody against the E envelope protein. As in all of the assays described above, rinsing can be performed to remove non-specifically bound material from the solid support.

使用本文揭示之方法時，已發現登革熱病毒會與CD14+巨噬細胞表面上之DVLR1/CLEC5A結合。參見實施例11。再者，已證明DVLR1/CLEC5A結合於登革熱病毒造成DAP12之活化，其最終導致促發炎性細胞激素TNF-α、MIP-1 α、IFN-α及IL-8從巨噬細胞之釋放。參見實施例12。此等細胞激素之釋放參與出血性登革熱(DHF)及登革熱休克症候群(DSS)之發展。When using the methods disclosed herein, it has been found that dengue virus binds to DVLR1/CLEC5A on the surface of CD14+ macrophages. See Example 11. Furthermore, binding of DVLR1/CLEC5A to dengue virus has been shown to cause activation of DAP12, which ultimately leads to the release of the pro-inflammatory cytokines TNF-α, MIP-1 α, IFN-α and IL-8 from macrophages. See Example 12. The release of these cytokines is involved in the development of hemorrhagic dengue fever (DHF) and dengue shock syndrome (DSS).

根據本文揭示方法之具體例，其已明確顯示DVLR1/CLEC5A可與登革熱病毒作用。參見實施例16-18。此外，其顯示DVLR1/CLEC5A可調節DAP12之磷酸化作用，咸信該磷酸化作用可至少部分調節諸如TNF-α之促發炎性細胞激素的釋出。參見實施例18。當經登革熱病毒感染之細胞中的DVLR1/CLEC5A表現下降(knock down)時，DAP12之磷酸化作用會降低，且包括TNF-α之促發炎性細胞激素之分泌會減少，同時並不會影響諸如干擾素-α之負責進行病毒清除之細胞激素的分泌。參見實施例18-19。根據具體例，DVLR1/CLEC5A之下降(knock down)可使用習知之RNA-干擾技術完成，包括si-RNA及sh-RNA兩者之使用。參見實施例18-19。According to a specific example of the method disclosed herein, it has been clearly shown that DVLR1/CLEC5A can interact with dengue virus. See Examples 16-18. Furthermore, it was shown that DVLR1/CLEC5A regulates the phosphorylation of DAP12, which is believed to at least partially regulate the release of pro-inflammatory cytokines such as TNF-[alpha]. See Example 18. When the DVLR1/CLEC5A expression in dengue virus-infected cells is knocked down, the phosphorylation of DAP12 is reduced, and the secretion of inflammatory cytokines including TNF-α is reduced, and does not affect such as Interferon-alpha is responsible for the secretion of cytokines for viral clearance. See Examples 18-19. According to a specific example, the knockdown of DVLR1/CLEC5A can be accomplished using conventional RNA-interference techniques, including the use of both si-RNA and sh-RNA. See Examples 18-19.

對於會與病原體交互作用之先天性免疫受體之類別(identity)之認知，繼而可被用於開發能調節先天性免疫受體之活性之藥劑。例如，可以獲取能活化所鑑定出之先天免疫性受體之調節劑，以加強對於特定病原體之免疫反應。若先天性免疫受體與特定多醣體組合物之交互作用對於身體有害(例如當病原體引起過度發炎時)，則可獲取能降低先天性免疫受體之活性之調節劑。例如可使用能封阻病原體結合於先天性免疫受體之藥劑(諸如抗體)，以防止該病原體感染所造成之不期望促發炎反應之發生。同樣地，若本發明之篩選方法揭露特定病原體(諸如病毒)利用先天性免疫受體而獲取進入細胞之管道，則封阻該病原體結合於先天性免疫受體之藥劑將會防止病原體進入細胞。The recognition of the identity of the innate immune receptor that interacts with the pathogen can then be used to develop agents that modulate the activity of the innate immune receptor. For example, modulators that activate the identified innate immune receptors can be obtained to enhance the immune response to a particular pathogen. If the interaction between the innate immune receptor and the particular polysaccharide composition is harmful to the body (for example, when the pathogen causes excessive inflammation), a modulator that reduces the activity of the innate immune receptor can be obtained. For example, an agent (such as an antibody) capable of blocking the binding of a pathogen to an innate immune receptor can be used to prevent the undesired inflammatory response caused by the infection of the pathogen. Similarly, if the screening method of the present invention reveals that a particular pathogen (such as a virus) utilizes an innate immune receptor to gain access to the cell, the agent that blocks the pathogen from binding to the innate immune receptor will prevent the pathogen from entering the cell.

根據本文揭示方法之具體例，其顯示投與能夠減少可用DVLR1/CLEC5A結合位點之干擾藥劑可增加經登革熱病毒感染小鼠之存活率。根據具體例，其顯示投與能夠干擾DVLR1/CLEC5A與DVLR1/CLEC5A配體結合之DVLR1/CLEC5A抗體可增加小鼠之存活率。參見實施例25。According to a specific example of the methods disclosed herein, it is shown that administration of an interfering agent capable of reducing the available DVLR1/CLEC5A binding site increases the survival rate of dengue virus-infected mice. According to a specific example, it was shown that administration of a DVLR1/CLEC5A antibody capable of interfering with the binding of DVLR1/CLEC5A to a DVLR1/CLEC5A ligand increased the survival rate of the mouse. See Example 25.

在另一系列具體例中，本發明之融合蛋白質被用於干擾或防止多醣體與細胞表面上之先天性免疫受體間之交互作用。在該系列之具體例中，融合蛋白質包含在細胞表面上表現之先天性免疫受體之碳水化合物識別區域。然後使該表現先天性免疫受體之細胞與融合蛋白質在活體內或試管中接觸，藉此融合蛋白質和多醣體競爭與先天性免疫受體之結合。In another series of specific examples, the fusion proteins of the invention are used to interfere with or prevent interaction between the polysaccharide and the innate immune receptor on the cell surface. In a specific example of this series, the fusion protein comprises a carbohydrate recognition region of an innate immune receptor expressed on the surface of the cell. The cells expressing the innate immune receptor are then contacted with the fusion protein in vivo or in a test tube, whereby the fusion protein and the polysaccharide compete for binding to the innate immune receptor.

若多醣體與細胞表面上之先天性免疫受體間之交互作用對於生物產生有害的作用，可將治療有效量之融合蛋白質以醫藥組合物之形式投與至生物，以防止或減輕該交互作用。所投與之融合蛋白質之異源多肽較佳不會與任何細胞表面受體結合。例如，異源多肽可包含IgG Fc之突變型，該突變型不會與細胞表面上之Fc受體結合。If the interaction between the polysaccharide and the innate immune receptor on the cell surface has a deleterious effect on the organism, a therapeutically effective amount of the fusion protein can be administered to the organism in the form of a pharmaceutical composition to prevent or mitigate the interaction. . The heterologous polypeptide to which the fusion protein is administered preferably does not bind to any cell surface receptor. For example, a heterologous polypeptide can comprise a mutant of IgG Fc that does not bind to an Fc receptor on the cell surface.

純化purification

在另一系列具體例中，融合蛋白質被用於至少部分純化或單離包含融合蛋白質之碳水化合物識別區域所識別之特異性碳水化合物成分之多醣體。舉例言之，可將融合蛋白質固定在固體支撐物上，並使疑似含有或已知含有多醣體組合物之組合物與固體支撐物接觸。若該組合物包含可與融合蛋白質之碳水化合物識別區域結合之多醣體，則該多醣體會與融合蛋白質結合。然後可沖洗固體支撐物以移除組合物之非特異性結合成分，而結合之多醣體可藉由解開與融合蛋白質之交互作用而溶析，然後將其收集。舉例言之，若融合蛋白質包含凝集素受體之碳水化合物識別區域，則可使用EDTA螯合Ca²⁺ 以解開交互作用。以此方式，可以從複合物混合物純化出特異性多醣體組合物。在較佳具體例中，其使用該方法純化單離自靈芝之多醣體。In another series of specific examples, the fusion protein is used to at least partially purify or isolate a polysaccharide comprising a specific carbohydrate component identified by the carbohydrate recognition region of the fusion protein. For example, the fusion protein can be immobilized on a solid support and the composition suspected of containing or known to contain the polysaccharide composition contacted with the solid support. If the composition comprises a polysaccharide that binds to the carbohydrate recognition region of the fusion protein, the polysaccharide will bind to the fusion protein. The solid support can then be rinsed to remove non-specific binding components of the composition, and the bound polysaccharide can be resolved by uncoupling interaction with the fusion protein and then collected. For example, if the fusion protein comprises a carbohydrate recognition region of a lectin receptor, EDTA can be used to sequester Ca ²⁺ to untie the interaction. In this way, a specific polysaccharide composition can be purified from the complex mixture. In a preferred embodiment, the method is used to purify polysaccharides isolated from Ganoderma lucidum.

就上述純化方法而言，該固體支撐物可包含例如結合有融合蛋白質之管柱。適當的管柱包括瓊脂糖(Sepharose)蛋白質A管柱，其中包含IgG以做為異源多肽之融合蛋白質可經由融合蛋白質之IgG區域與蛋白質A之交互作用而結合在該管柱上。或者，可以經CNBr活化之管柱介質而與融合蛋白質結合。For the above purification methods, the solid support may comprise, for example, a column to which a fusion protein is bound. Suitable columns include a Sepharose Protein A column in which a fusion protein comprising IgG as a heterologous polypeptide can be bound to the column via interaction of the IgG region of the fusion protein with Protein A. Alternatively, the fusion protein can be bound via a CNBr activated column medium.

本發明亦提供可用於上述方法之任一者之套組。在一具體例中，套組包含本發明之融合蛋白質，其裝在一個或多個容器中。該套組亦可包含第二試劑，諸如與融合蛋白質之異源多肽區域特異性結合之抗體，舉例言之，若異源多肽為IgG或其片段，該第二試劑為抗-IgG抗體。該套組亦可包含檢測融合蛋白質與多醣體結合用之試劑及緩衝液。例如，在使用綴合有HRP之二次抗體檢測融合蛋白質與多醣體之結合之具體例中，該套組可包含建立增強之化學發光反應所需之試劑，例如一個或一個以上容器包含魯米諾(luminol)、對-香豆酸(p-coumaric acid)、Tris緩衝液及過氧化氫。該套組亦可包含一個或一個以上陽性對照多醣體。該套組亦可包含一個或一個以上用於上述方法之固體支撐物，例如，一個或一個以上PVDF膜或者一個或一個以上多孔微量滴定平皿。The invention also provides kits that can be used in any of the above methods. In one embodiment, the kit comprises a fusion protein of the invention contained in one or more containers. The kit may also comprise a second agent, such as an antibody that specifically binds to a heterologous polypeptide region of the fusion protein, for example, if the heterologous polypeptide is IgG or a fragment thereof, the second agent is an anti-IgG antibody. The kit may also include reagents and buffers for detecting binding of the fusion protein to the polysaccharide. For example, in a specific example of detecting binding of a fusion protein to a polysaccharide using a secondary antibody conjugated with HRP, the kit may comprise reagents required to establish an enhanced chemiluminescent reaction, for example one or more containers comprising rumii Luminol, p-coumaric acid, Tris buffer, and hydrogen peroxide. The kit may also contain one or more positive control polysaccharides. The kit may also comprise one or more solid supports for use in the above methods, for example, one or more PVDF membranes or one or more porous microtiter plates.

調節劑Conditioner

如上述，本發明之方法鑑定出會與特定多醣體交互作用之先天性免疫受體。該等資料使吾等得以得到所鑑定出之先天性免疫受體之調節劑。調節劑可為先天性免疫受體之催動劑、桔抗劑(包括競爭性或非競爭性桔抗劑)或逆向催動劑。調節劑可(但非限於此)抑制多醣體與先天性免疫受體之結合；加強多醣體與先天性免疫受體之結合；或者做為可與先天性免疫受體結合之多醣體之擬似劑，藉此甚至在缺乏多醣體時亦可活化先天性免疫受體。As described above, the method of the present invention identifies an innate immune receptor that interacts with a particular polysaccharide. This information allows us to obtain the modulators of the identified innate immune receptors. The modulator may be a promoter of an innate immune receptor, an orange antagonist (including a competitive or non-competitive orange antagonist) or a retrograde agonist. The modulator may, but is not limited to, inhibit binding of the polysaccharide to the innate immune receptor; enhance binding of the polysaccharide to the innate immune receptor; or act as a mimetic of the polysaccharide that binds to the innate immune receptor Thereby, the innate immune receptor can be activated even in the absence of polysaccharides.

先天性免疫受體之調節劑包括抗體。舉例言之，對抗先天性免疫受體之拮抗性抗體可防止病原體與先天性免疫受體之結合。在一些情況，該抗體為中和抗體，因其會防止病原體進入表現該先天性免疫受體之細胞。或者，催動性抗體可做為對於細胞施予有益作用之多醣體組合物之擬似物。拮抗性抗體與先天性免疫受體之結合方式亦可封阻受體於病原體結合時所進行之下游信號傳導。抗體可為(但非限於此)多株抗體、單株抗體、單價抗體、雙特異性抗體、雜綴合型抗體、多特異性抗體、人類抗體、人型化抗體或嵌合抗體；單鏈抗體；Fab片段、F(ab')片段、由Fab表現庫產生之片段；抗基因型(anti-idiotypic，anti-Id)抗體；以及上述抗體之任一種之抗原決定部位-結合性片段。本文所用之術語「抗體」係指免疫球蛋白分子及免疫球蛋白分子之免疫活性部分，亦即含有可與抗原免疫特異性地結合之抗原結合部位之分子。該免疫球蛋白分子可為免疫球蛋白分子之任何類型(例如IgG、IgE、IgM、IgD、IgA及IgY)、類別(例如、IgG1、IgG2、IgG3、IgG4、IgA1及IgA2)或亞類。再者，術語「抗體」(Ab)或「單株抗體」(Mab)意欲包括完整的分子以及能與蛋白質特異性地結合之抗體片段(諸如Fab及F(ab')₂ 片段)。Fab及F(ab')₂ 片段缺乏完整抗體之Fc片段，因而可較快速地從動物或植物之循環清除，且其與完整的抗體相較，具有較少的非特異性組織結合性(Waahl et al.、J.Nucl.Med.24：316-325(1983))。製造抗體催動劑之方法例如記載於PCT公開號WO 96/40281；美國專利第5,811,097號；Deng et al.,Blood 92(6)：1981-1988(1998)；Chen et al.,Cancer Res.58(16)：3668-3678(1998)；Harrop et al.,J.Immunol.161(4)：1786-1794(1998)；Zhu et al.,Cancer Res.58(15)：3209-3214(1998)；Yoon et al.,J.Immunol.160(7)：3170-3179(1998)；Prat et al.,J.Cell.Sci.111(Pt2)：237-247(1998)；Pitard et al.,J.Immunol.Methods 205(2)：177-190(1997)；Liautard et al.、Cytokine 9(4)：233-241(1997)；Carlson et al.,J.Biol.Chem.272(17)：11295-11301(1997)；Taryman et al.,Neuron 14(4)：755-762(1995)；Muller et al.,Structure 6(9)：1153-1167(1998)；Bartunek et al.,Cytokine 8(1)：14-20(1996)；Harlow et al.,Antibodies：A Laboratory Manual,(Cold Spring Harbor Laboratory Press,2nd ed.1988)；Hammerling,et al.,in：Monoclonal Antibodies and T-Cell Hybridomas 563-681(Elsevier,N.Y.,1981)(所有文獻全文以參考資料之方式納入本文)。Modulators of innate immune receptors include antibodies. For example, antagonistic antibodies against innate immune receptors prevent the binding of pathogens to innate immune receptors. In some cases, the antibody is a neutralizing antibody as it prevents the pathogen from entering cells expressing the innate immune receptor. Alternatively, the priming antibody can be used as a mimetic of a polysaccharide composition that imparts a beneficial effect on the cells. The binding of the antagonist antibody to the innate immune receptor also blocks downstream signaling by the receptor when it binds to the pathogen. The antibody may be, but is not limited to, a plurality of antibodies, a monoclonal antibody, a monovalent antibody, a bispecific antibody, a heteroconjugate antibody, a multispecific antibody, a human antibody, a humanized antibody or a chimeric antibody; An antibody; a Fab fragment, an F(ab') fragment, a fragment produced by a Fab expression library; an anti-idiotypic (anti-id) antibody; and an epitope-binding fragment of any of the above antibodies. The term "antibody" as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that immunospecifically binds to an antigen. The immunoglobulin molecule can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules. Furthermore, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is intended to include intact molecules as well as antibody fragments (such as Fab and F(ab') ₂ fragments) that specifically bind to proteins. The Fab and F(ab') ₂ fragments lack the Fc fragment of the intact antibody and thus can be cleared from the animal or plant cycle more rapidly, and have less non-specific tissue binding than the intact antibody (Waahl Et al., J. Nucl. Med. 24: 316-325 (1983)). Methods of making antibody motivators are described, for example, in PCT Publication No. WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6): 1981-1988 (1998); Chen et al., Cancer Res. 58(16): 3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58(15): 3209-3214 ( 1998); Yoon et al., J. Immunol. 160(7): 3170-3179 (1998); Prat et al., J. Cell. Sci. 111 (Pt 2): 237-247 (1998); Pitard et al J. Immunol. Methods 205(2): 177-190 (1997); Liautard et al., Cytokine 9(4): 233-241 (1997); Carlson et al., J. Biol. Chem. 272 ( 17): 11295-11301 (1997); Taryman et al., Neuron 14(4): 755-762 (1995); Muller et al., Structure 6(9): 1153-1167 (1998); Bartunek et al. , Cytokine 8(1): 14-20 (1996); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T - Cell Hybridomas 563-681 (Elsevier, NY, 1981) (all references are incorporated herein by reference).

本發明提供可在DV感染後防止TNF-α從巨噬細胞釋出之抗-DVLR1/CLEC5A單株抗體之非限定特異性實施例。參見實施例15。此等抗體可用於本文所指定之醫藥組合物及治療方法，尤其是用於治療或預防人類DV感染之組合物及方法。The present invention provides a non-limiting specific embodiment of an anti-DVLR1/CLEC5A monoclonal antibody that prevents the release of TNF-[alpha] from macrophages following DV infection. See Example 15. Such antibodies can be used in the pharmaceutical compositions and methods of treatment as specified herein, particularly compositions and methods for treating or preventing human DV infection.

本發明亦提供可在DV或JEV感染後防止TNF-α從巨噬細胞釋出之人型化抗-DVLR1/CLEC5A抗體。參見實施例20-26。在特定具體例中，該人型化抗體係選自由人型化抗體9B12、3E12A2、3E12C1、3E12G9、及8H8F5所組成之群。此等抗體可用於本文所指定之醫藥組合物及治療方法，尤其是用於治療或預防人類DV感染之組合物及方法。特定之治療包括抑制DV-誘發性血漿滲漏以及皮下及生命器官(vital organ)出血。該等人型化抗體可作為DV-誘發性出血休克及敗血症之治療方法。尤被考量者，本文所述有關由病毒所造成之細胞激素刺激減輕可應用於所有可結合並調節細胞之刺激性受體的病毒。The invention also provides a humanized anti-DVLR1/CLEC5A antibody that prevents the release of TNF-[alpha] from macrophages following infection with DV or JEV. See Examples 20-26. In a specific embodiment, the humanized anti-system is selected from the group consisting of humanized antibodies 9B12, 3E12A2, 3E12C1, 3E12G9, and 8H8F5. Such antibodies can be used in the pharmaceutical compositions and methods of treatment as specified herein, particularly compositions and methods for treating or preventing human DV infection. Specific treatments include inhibition of DV-induced plasma leakage and subcutaneous and vital organ bleeding. These humanized antibodies can be used as a treatment for DV-induced hemorrhagic shock and sepsis. In particular, the cytokine stimuli alleviated by viruses described herein can be applied to all viruses that bind to and modulate the stimulatory receptors of cells.

再者，病毒之發現及治療原則可以類似方式延伸至細胞侵入受體以及細菌、真菌、及寄生物之作用。本發明之方法將可使一般精於本技術者能夠判斷病原體之結合特性(亦即，其與何種受體結合)，判斷與該受體之結合具有何種作用，並提供諸如抗體之干擾劑以干擾病原體結合目標受體之能力。Furthermore, the discovery and treatment principles of the virus can be extended in a similar manner to cellular invasion of receptors as well as bacteria, fungi, and parasites. The method of the present invention will enable a person skilled in the art to determine the binding properties of a pathogen (i.e., which receptor it binds to), determine the role of binding to the receptor, and provide interference such as antibodies. The agent interferes with the ability of the pathogen to bind to the target receptor.

根據具體例，藉由融合小鼠脾細胞及NS1骨髓配對細胞而產生之單株抗體(mAbs)抗-DVLR1/CLEC5A mAb可以噬菌體展示技術產生，以產生單鏈人類抗-人類DVLR1/CLEC5A mAbs。催動性及拮抗性mAbs可根據實例19-25所揭示之篩選方法而選擇。According to a specific example, monoclonal antibodies (mAbs) anti-DVLR1/CLEC5A mAbs produced by fusion of mouse spleen cells and NS1 bone marrow pairing cells can be produced by phage display technology to produce single-stranded human anti-human DVLR1/CLEC5A mAbs. The motility and antagonist mAbs can be selected according to the screening methods disclosed in Examples 19-25.

為降低現有小鼠抗-人類DVLR1/CLEC5A mAbs之抗原性，根據具體例，以人類免疫球蛋白G1(IgG1)取代野生型Fc部分。為進一步廢除Fc與Fc受體之結合並防止補體活化，可使用人類IgG1之突變Fc片段(L234A、L235E、G237A、及P331S)取代野生型Fc部分以產生人型化mAbs。為進一步降低抗原性，可以人類序列取代抗體V區域之框架區域。To reduce the antigenicity of existing mouse anti-human DVLR1/CLEC5A mAbs, the wild-type Fc portion was replaced with human immunoglobulin G1 (IgG1) according to a specific example. To further abolish the binding of Fc to the Fc receptor and prevent complement activation, the wild-type Fc portion can be replaced with a mutant Fc fragment of human IgG1 (L234A, L235E, G237A, and P331S) to produce humanized mAbs. To further reduce antigenicity, the framework region of the antibody V region can be replaced by a human sequence.

先天性免疫受體之調節劑亦包括由高通量篩選方法所鑑定出之小分子。該高通量篩選方法典型地提供含有大量潛在治療化合物(例如配體或調節劑化合物)之組合化學品或肽庫。然後在一個或一個以上檢定分析中篩選該等組合化學品庫或配位體庫，以鑑定出此等可與所探究之先天性免疫受體結合之庫成員(例如特定的化學品種類或亞類)。如此鑑定出之化合物可做為習知之引導化合物(lead compound)，或者本身可做為潛在的或實際的治療劑。Modulators of innate immune receptors also include small molecules identified by high throughput screening methods. This high throughput screening method typically provides a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (eg, ligand or modulator compounds). These combinatorial chemical libraries or library of ligands are then screened in one or more assays to identify such library members that can bind to the innate immune receptors being explored (eg, specific chemical species or sub- class). The compound thus identified may be used as a conventional lead compound or may itself be a potential or actual therapeutic agent.

組合化學品庫為藉由化學合成或生物合成，或者藉由組合許多化學建構基塊(即試劑，諸如胺基酸)所產生之在化學上分歧之化合物之集合物。舉例言之，線性組合庫，例如多肽或肽庫，藉由以對既定化合物長度(即在多肽或肽化合物中胺基酸之數目)而言每種可能的方式，組合一套化學建構基塊而形成。數以百萬計之化合物可經由組混化學建構基塊而合成。A combinatorial chemical library is a collection of chemically distinct compounds produced by chemical synthesis or biosynthesis, or by combining a number of chemical building blocks (ie, reagents such as amino acids). For example, a linear combinatorial library, such as a polypeptide or peptide library, combines a set of chemical building blocks by each possible means for a given compound length (ie, the number of amino acids in a polypeptide or peptide compound). And formed. Millions of compounds can be synthesized by building chemical blocks.

組合化學品庫之製備及篩選為精於相關技術者所熟知。該組合庫包括，但非限於肽庫(例如美國專利第5,010,175號；Furka,1991,Int.J.Pept.Prot.Res.,37,487-493；以及Houghton et al.,1991,Nature,354：84-88)。亦可使用其他產生化學上分歧之化學品庫之化學方法。化學上分歧之化學品庫之化學類別之非限定性例子包括肽(PCT公開號WO 91/019735)，經編碼之肽(PCT公開號WO 93/20242)，隨機生物寡聚物(PCT公開號WO 92/00091)，苯并二吖呯類(benzodiazepines)(美國專利第5,288,514號)，分歧異構物(diversomers)諸如海因類(hydantoins)、苯并二 呯類(benzodiazepines)及二肽類(Hobbs et al.,1993,Proc.Natl.Acad.Sci.USA,90：6909-6913)，類乙烯多肽(Hagihara et al.,1992,J.Amer.Chem.Soc.,114：6568)，具葡萄糖框架(glucose scaffolding)之非肽類擬肽(Hirschmann et al.,1992,J.Amer.Chem.Soc.,114：9217-9218)，小型化合物庫(Chen et al.,1994,J.Amer.Chem.Soc.,116：2661)，寡聚胺基甲酸酯(Cho et al.,1993,Science,261：1303)，及/或膦酸肽酯(Campbell et al.,1994,J.Org.Chem.,59：658)之類似有機合成，核酸庫(例如參見美國專利第5,270,163號，其述及核酸配體(亦稱為“aptamers”)之產生)，肽-核酸庫(美國專利第5,539,083號)，抗體庫(例如Vaughn et al.,1996,Nature Biotechnology,14(3)：309-314)及PCT/US96/10287)，碳水化合物庫(例如參見Liang et al.,1996,Science,274-1520-1522)及美國專利第5,593,853號)，小型有機分子庫(例如，苯并二呯類，Baum C&EN,Jan.18,1993,page 33；以及美國專利第5,288,514號；類異戊二烯類，美國專利第5,569,588號；噻唑啶酮類(thiazolidinones)及間全氫噻口井酮類(metathiazanones)，美國專利第5,549,974號；吡咯啶類(pyrrolidines)，美國專利第5,525,735號及第5,519,134號；嗎啉類化合物，美國專利第5,506,337號；以及類似者)。The preparation and screening of combinatorial chemical libraries is well known to those skilled in the relevant art. Such combinatorial libraries include, but are not limited to, peptide libraries (e.g., U.S. Patent No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37, 487-493; and Houghton et al., 1991, Nature, 354:84). -88). Other chemical methods that produce chemically distinct chemical libraries can also be used. Non-limiting examples of chemical classes of chemically distinct chemical libraries include peptides (PCT Publication No. WO 91/019735), encoded peptides (PCT Publication No. WO 93/20242), random biological oligomers (PCT Publication Number) WO 92/00091), benzodiazepines (U.S. Patent No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 6909-6913), an ethylene-like polypeptide (Hagihara et al., 1992, J. Amer. Chem. Soc., 114: 6568), Non-peptide peptoids with glucose scaffolding (Hirschmann et al., 1992, J. Amer. Chem. Soc., 114: 9217-9218), small compound library (Chen et al., 1994, J.). Amer. Chem. Soc., 116: 2661), oligomeric urethane (Cho et al., 1993, Science, 261:1303), and/or phosphonate peptide ester (Campbell et al., 1994, J). .Org. Chem., 59: 658), similar to organic synthesis, nucleic acid libraries (see, for example, U.S. Patent No. 5,270,163, which describes the production of nucleic acid ligands (also known as "aptamers"), peptide-nucleic acid libraries (USA) Patent No. 5,539,083), antibody library (eg Vaug Hn et al., 1996, Nature Biotechnology, 14(3): 309-314) and PCT/US96/10287), carbohydrate libraries (see, for example, Liang et al., 1996, Science, 274-1520-1522) and the United States Patent No. 5,593,853), a small organic molecular library (for example, benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S. Patent No. 5,288,514; isoprenoids, U.S. Patent No. 5,569,588 ; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; Morpholine Compounds, USA Patent No. 5,506,337; and the like).

製備組合庫之裝置可從市面購得(例如357 MPS,390 MPS,Advanced Chem Tech,Louisville Ky.；Symphony,Rainin,Woburn,Mass.；433A Applied Biosystems,Foster City,Calif.；9050 Plus,Millipore,Bedford,Mass.)。此外，許多組合庫可從市面購得(例如,ComGenex,Princeton,N.J.；Asinex,Moscow,Russia；Tripos,Inc.,St.Louis,Mo.；ChemStar,Ltd.,Moscow,Russia；3D Pharmaceuticals,Exton,Pa.；Martek Biosciences,Columbia,Md.,以及類似者)。Devices for preparing combinatorial libraries are commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, many combinatorial libraries are commercially available (eg, ComGenex, Princeton, NJ; Asinex, Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton , Pa.; Martek Biosciences, Columbia, Md., and the like).

醫藥組合物Pharmaceutical composition

本發明亦提供醫藥組合物。在一些具體例中，醫藥組合物包含本發明之融合蛋白質。在其他具體例中，醫藥組合物包含先天性免疫受體之調節劑(例如對抗先天性免疫受體諸如DVLR1/CLEC5A之抗體，包括實施例15所例示之抗體)。在該等醫藥組合物中，融合蛋白質或先天性免疫受體調節劑構成「活性化合物」。在一些具體例中，其將醫藥組合物投與至病患，以治療或預防以多醣體結合至病患細胞表面之先天性免疫受體為特徵之疾病或失調。在其他具體例中，其係於增加先天性免疫受體之活性對於病患有助益之情況，將該等醫藥組合物投與至病患以活化該先天性免疫受體。在另一些具體例中，其將醫藥組合物投與至病患，以加強多醣體組合物與先天性免疫受體之結合。The invention also provides pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises a fusion protein of the invention. In other embodiments, the pharmaceutical composition comprises a modulator of an innate immune receptor (eg, an antibody against an innate immune receptor such as DVLR1/CLEC5A, including the antibody exemplified in Example 15). In such pharmaceutical compositions, a fusion protein or an innate immune receptor modulator constitutes an "active compound." In some embodiments, the pharmaceutical composition is administered to a patient to treat or prevent a disease or disorder characterized by an innate immune receptor that binds to the surface of the patient's cell. In other embodiments, the pharmaceutical composition is administered to a patient to activate the innate immune receptor by increasing the activity of the innate immune receptor for the benefit of the patient. In other embodiments, the pharmaceutical composition is administered to a patient to enhance binding of the polysaccharide composition to an innate immune receptor.

醫藥組合物除了包含活性化合物之外，較佳復包含至少一種醫藥上可接受之載劑。本文所用之術語「醫藥上可接受之載劑」包括與醫藥投與相容之溶劑、分散體介質、包衣、抗細菌劑及抗真菌劑、等張劑及吸收延遲劑以及類似物。亦可將輔助的活性化合物納入組合物中。將醫藥組合物調配成與其意欲的投與途徑相容。投與途徑之例子包括腸道外投與，例如靜脈內、皮內、皮下、經口(例如吸入)、穿皮(外用)、穿黏膜及直腸投與。用於腸道外、皮內或皮下投與之溶液或懸浮液可包括下列成分：無菌稀釋液諸如注射用水、鹽水溶液、固定油、聚乙二醇、甘油、丙二醇或其他合成溶劑；抗細菌劑諸如苄醇或對羥基苯甲酸甲酯；抗氧化劑諸如抗壞血酸或亞硫酸氫鈉；螯合劑諸如乙二胺四乙酸；緩衝液諸如乙酸鹽、檸檬酸鹽或磷酸鹽；以及等張性調整劑諸如氯化鈉或葡萄糖。pH值可用酸或鹼諸如鹽酸或氫氧化鈉調整。腸道外製劑可密封在玻璃或塑膠製之安瓿、可拋棄式注射筒或多劑量小瓶中。The pharmaceutical composition preferably comprises, in addition to the active compound, at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. Supplementary active compounds can also be included in the compositions. The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following ingredients: sterile diluents such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents Such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium hydrogen sulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate or phosphate; and an isotonicity adjusting agent such as Sodium chloride or glucose. The pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

本文中所用之術語「病患」係指人類及非人類靈長類(例如猩猩、獼猴、狨猴)，家畜(例如綿羊、牛、馬、驢子、豬)，寵物(例如狗、貓)，實驗室試驗動物(例如小鼠、兔子、大鼠、天竺鼠、倉鼠)，圈養之野生動物(例如狐狸、鹿)以及任何可從本發明藥劑獲益之其他生物。對於可從本文所述之藥劑獲益之動物類型沒有限制。在本發明中最佳之病患為人類。不論是人類或非人類生物皆可被稱為病患(patient)、個體(individual)、動物、宿主(host)或接受者(recipient)。The term "patient" as used herein refers to humans and non-human primates (eg, orangutans, macaques, simians), livestock (eg, sheep, cattle, horses, scorpions, pigs), pets (eg, dogs, cats), Laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), captive wild animals (e.g., foxes, deer), and any other organisms that can benefit from the agents of the present invention. There are no restrictions on the types of animals that can benefit from the agents described herein. The best patient in the present invention is a human. Whether human or non-human organisms can be referred to as a patient, an individual, an animal, a host, or a recipient.

適合注射用之醫藥組合物包括無菌水溶液(在可溶於水之情況)或分散體以及無菌注射溶液或分散液之即時調配製劑用之無菌粉末。就靜脈內投與而言，適當的載劑包括生理食鹽水、抑菌水、Cremophor EL.TM.(BASF,Parsippany,N.J.)或磷酸鹽緩衝鹽水(PBS)。在所有情況，組合物必須為無菌以及具有某種程度之流體性，以易於注射。組合物在製造及貯存條件下應當安定且必須對抗微生物諸如細菌及真菌之污染作用而進行防腐。載劑可為溶劑或分散體介質，其含有例如水、乙醇、多元醇(例如甘油、丙二醇及液體聚乙二醇以及類似物)以及其適當的混合物。適當的流體性例如可以藉由使用塗膜諸如卵磷脂而維持，在分散體之情況藉由保持所需之粒度而維持以及藉由使用界面活性劑而維持。防止微生物之作用可藉由各種抗細菌劑及抗真菌劑，例如，對羥基苯甲酸酯、氯丁醇、酚、抗壞血酸、乙汞硫柳酸鈉(thimerosal)及類似物而達成。在許多情況，組合物較佳包括等張劑，例如，糖、多元醇諸如甘露醇及山梨醇、或氯化鈉。可注射組合物之延長吸收可藉由在組合物中包括延遲吸收之藥劑例如單硬脂酸鋁及明膠而達成。The pharmaceutical compositions suitable for injectable use include sterile powders in the form of a sterile aqueous solution (in the case of water) or dispersions and sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and have some degree of fluidity for ease of injection. The composition should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating film such as lecithin, by the maintenance of the desired particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, the compositions preferably include an isotonic agent, for example, a sugar, a polyhydric alcohol such as mannitol and sorbitol, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin, in the compositions.

無菌注射溶液可藉由將所需量之活性化合物引進適當的溶劑中，視需要同時引進上文列舉之成分之一種或組合物，繼而過濾滅菌而製備。一般而言，分散液藉由將活性化合物引進無菌載劑而製備，該無菌載劑含有基本分散介質及上文列舉者中之所需其他成分。製備無菌注射溶液所用之無菌粉末之情況，較佳之製備方法為真空乾燥及冷凍乾燥，其可從先前經無菌過濾之溶液得到活性成分加上任何其他期望成分之粉末。Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent, optionally incorporating one or a combination of the ingredients listed above, followed by filtration sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a base dispersion medium and other ingredients in the compositions listed above. In the case of a sterile powder for the preparation of a sterile injectable solution, the preparation is preferably vacuum drying and lyophilization, which yields a powder of the active ingredient plus any other desired ingredient from a previously sterilely filtered solution.

口服組合物通常包括惰性稀釋劑或可食用之載劑。就口服治療投與之目的而言，活性化合物可與賦形劑混合並以錠劑、含錠或膠囊劑(例如明膠膠囊劑)之形式使用。口服組合物亦可用流體載劑製備，以做為漱口水。醫藥相容性結合劑或佐劑物質可被納入做為組合物之一部分。錠劑、丸劑、膠囊劑、含錠劑及類似製劑可含有下列成分之任一種或具有相似性質之化合物：黏合劑諸如微晶形纖維素、西黃耆膠或明膠；賦形劑諸如澱粉或半乳糖；崩散劑諸如藻膠酸、Primogel或玉米澱粉；潤滑劑諸如硬脂酸鎂或Sterotes；滑劑諸如膠體二氧化矽；甜味劑諸如蔗糖或糖精；或調味劑諸如薄荷(peppermint)、水楊酸甲酯或橙類調味劑。Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound may be mixed with excipients and employed in the form of lozenges, lozenges or capsules (for example, gelatin capsules). Oral compositions can also be prepared with a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges and the like may contain any of the following ingredients or compounds having similar properties: binders such as microcrystalline cellulose, tragacanth or gelatin; excipients such as starch or Galactose; disintegrating agents such as alginate, Primogel or corn starch; lubricants such as magnesium stearate or Sterotes; slip agents such as colloidal cerium oxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, Methyl salicylate or orange flavoring.

就藉由吸入之投與而言，化合物以氣溶膠噴霧劑(aerosol spray)之形式從含有適當推進劑(例如氣體諸如二氧化碳)之加壓容器或分配器輸出，或由霧化器(nebulizer)輸出。In the case of administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas such as carbon dioxide, or by a nebulizer. Output.

全身性投與亦可經由穿過黏膜(transmucosal)或穿皮投與。就穿過黏膜或穿皮投與而言，在調配物中使用適於透過障壁之滲透劑。此等滲透劑在本技術中為眾所週知，例如就穿過黏膜投與而言，包括清潔劑、膽鹽及褐黴酸(fusidic acid)衍生物。穿過黏膜投與可經由使用鼻噴霧劑或栓劑而達成。就穿皮投與而言，活性化合物被調配成本技術所週知之軟膏、油膏、凝膠或乳霜。Systemic administration can also be administered via transmucosal or transdermal. For administration through the mucosa or through the skin, a penetrant suitable for passage through the barrier is used in the formulation. Such penetrants are well known in the art, for example, in the case of transmucosal administration, including detergents, bile salts, and fusidic acid derivatives. Administration through the mucosa can be achieved via the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels or creams which are well known in the art.

該等化合物亦可被製成供直腸輸送用之栓劑(例如使用習用之栓劑基劑諸如可可油脂及其他甘油酯)或滯留性灌腸劑。The compounds may also be formulated as suppositories for rectal delivery (for example, using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

在一具體例中，活性化合物藉由使用能保護該化合物使其免於從身體快速清除之載劑，而製備成例如控制釋放調配物，該控制釋放調配物包括植入物、顯微包膠輸送系統。可以使用生物可降解、生物相容性聚合物諸如乙烯-乙酸乙烯酯共聚物、聚酸酐、聚羥乙酸、膠原、聚原酯(polyortho-ester)及聚乳酸。該等調配物之製法對精於本技術者而言為顯而易知。該等物質亦可從Alza公司及Nova藥品公司獲得。微脂粒懸浮液(包括具有針對細胞-特異性抗原之單株抗體而可標定於感染細胞之微脂粒)亦可被用做醫藥上可接受之載劑。此等微脂粒懸浮液可依照精於本技術者已知之方法製備，例如美國專利第4,522,811號所述者。In one embodiment, the active compound is prepared, for example, as a controlled release formulation by using a carrier that protects the compound from rapid elimination from the body, the controlled release formulation including implants, microencapsulation Conveyor system. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyortho-esters, and polylactic acid can be used. The preparation of such formulations is readily apparent to those skilled in the art. These substances are also available from Alza and Nova Pharmaceuticals. A liposome suspension (including vesicles identifiable to infected cells with a monoclonal antibody directed against a cell-specific antigen) can also be used as a pharmaceutically acceptable carrier. Such vesicle suspensions can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

將口服或腸道外組合物調配成單位劑型係為有利，因為便於投與及劑量均勻。本文所稱之「單位劑型」係指適於做為待治療病人之單位劑量之物理上分離之單位；各單位含有經計算能產生期望治療作用之預定量活性化合物以及所需之醫藥載劑。It is advantageous to formulate the oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. "Unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for the patient to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, and the required pharmaceutical carrier.

該等化合物之毒性及治療效力可在細胞培養物或實驗動物中藉由標準醫藥方法測定，例如測定LD50(使群體之50%死亡之劑量)以及ED50(在50%群體中治療有效之劑量)。毒性作用與治療作用間之劑量比稱為治療指數且係以LD50/ED50比表示。以顯示高治療指數之化合物為較佳。雖然可以使用顯示毒性副作用之化合物，但應小心設計將該等化合物標定於受犯組織部位之輸送系統，以使對於未感染細胞之潛在傷害減至最低，藉此減少副作用。The toxicity and therapeutic efficacy of such compounds can be determined in cell culture or laboratory animals by standard pharmaceutical methods, such as determining LD50 (a dose that causes 50% of the population to die) and ED50 (a therapeutically effective dose in 50% of the population). . The dose ratio between toxic and therapeutic effects is called the therapeutic index and is expressed as the LD50/ED50 ratio. A compound exhibiting a high therapeutic index is preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design such compounds to be targeted to the delivery system of the affected tissue site to minimize potential damage to uninfected cells, thereby reducing side effects.

從細胞培養物檢定分析及動物研究得到之數據可用於調配某範圍之劑量以供病患使用。該等化合物之劑量較佳在包括ED50且引起極微或未引起毒性之循環濃度範圍內。劑量可在該範圍內，視所用劑型及投與途徑而變化。對於本發明方法所用之任何化合物而言，治療有效劑量初始可從細胞培養物檢定分析評估。在動物模型中調製能達到包括IC50(即受試化合物達到症狀之最大抑制之一半之濃度，該IC50如在細胞培養物中測得者)之循環血漿濃度範圍的劑量。該資料可被用於更精確地測定在病患中之有用劑量。血漿濃度例如可藉由高效液相層析測量。Data from cell culture assays and animal studies can be used to formulate a range of doses for use by patients. The dosage of such compounds is preferably within a range of circulating concentrations that include ED50 and cause little or no toxicity. The dosage may be within this range, depending on the dosage form employed and the route of administration. For any compound used in the methods of the invention, the therapeutically effective dose can be initially assessed from cell culture assays. Modulations in the animal model are made to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration at which the test compound achieves one-half of the maximum inhibition of symptoms, as measured in cell culture). This data can be used to more accurately determine the useful dose in a patient. The plasma concentration can be measured, for example, by high performance liquid chromatography.

如在本文中所定義，本發明之活性化合物之治療有效量可為約0.001至30毫克/公斤體重，較佳約0.01至25毫克/公斤體重，更佳約0.1至20毫克/公斤體重，甚至更佳約1至10毫克/公斤，2至9毫克/公斤，3至8毫克/公斤，4至7毫克/公斤，或者5至6毫克/公斤體重。活性化合物可以每星期1次至每天3次或3次以上之頻率投與，總計投與約1至10星期，較佳2至8星期，更佳約3至7星期，甚至更佳約4、5或6星期，但非限於此。精於本技術人士當了解某些因子會影響有效治療病患所需之劑量及給藥時間，該等因子包括，但不限於，疾病或失調之嚴重度、先前之治療、病患之一般健康狀況或年齡以及其他存在之疾病。再者，病患以治療有效量之本發明醫藥組合物之治療可包括單一治療或較佳包括一系列之治療。A therapeutically effective amount of an active compound of the invention, as defined herein, may range from about 0.001 to 30 mg/kg body weight, preferably from about 0.01 to 25 mg/kg body weight, more preferably from about 0.1 to 20 mg/kg body weight, or even More preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The active compound can be administered from once a week to three times a day or three times or more, for a total of about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, even more preferably about 4. 5 or 6 weeks, but not limited to this. Those skilled in the art will understand that certain factors affect the dosage and time of administration required to effectively treat a patient, including, but not limited to, the severity of the disease or disorder, prior treatment, general health of the patient. Condition or age and other existing diseases. Furthermore, the treatment of a pharmaceutical composition of the invention in a therapeutically effective amount may include a single treatment or preferably a series of treatments.

基因治療及RNAiGene therapy and RNAi

編碼本發明之融合蛋白質之構築體可被用作基因治療規程之一部分，以將治療有效劑量之受體融合蛋白質輸送至病患。在活體中將核酸引進細胞之較佳途徑為藉由使用含有編碼本發明之融合蛋白質之核酸之病毒載體。以病毒載體感染細胞具有下述優點：高比例之標定細胞可接受核酸。此外，病毒載體中所編碼之分子，例如藉由病毒載體所含之cDNA所編碼之分子，在攝入病毒載體核酸之細胞中能有效率地表現。Constructs encoding the fusion proteins of the invention can be used as part of a gene therapy protocol to deliver a therapeutically effective amount of a receptor fusion protein to a patient. A preferred route for introducing a nucleic acid into a cell in a living body is by using a viral vector containing a nucleic acid encoding the fusion protein of the present invention. Infecting cells with a viral vector has the advantage that a high proportion of the labeled cells can accept nucleic acids. Furthermore, the molecule encoded in the viral vector, for example, the molecule encoded by the cDNA contained in the viral vector, can be efficiently expressed in cells ingesting the viral vector nucleic acid.

反錄病毒(retrovirus vectors)及腺病毒相關性病毒載體可做為在活體中移轉編碼融合蛋白質之外源核酸分子之重組基因輸送系統。此等載體將核酸有效率地輸送入細胞中且所移轉之核酸被安定地整合入宿主之染色體DNA中。只製造複製-缺陷型反錄病毒之特異化細胞系(稱為「套裝細胞」)之開發使反錄病毒在基因治療之利用性上更為增加，且缺陷型反錄病毒經特徵定性而應用於基因治療目的之基因轉移中(該法之評介可參考Miller,A.D.(1990)Blood 76：271)。複製用缺陷型病毒可被組裝入病毒粒子中，該病毒粒子可藉由標準技術及使用輔助病毒感染標定細胞。產生重組反錄病毒之規程以及在試管中或活體中用該等病毒感染細胞之規程可記載於分子生物學之現有規程(Current Protocols in Molecular Biology,Ausubel,F.M.et al.,(eds.)Greene Publishing Associates,(1989),Sections 9.10-9.14)以及其他標準實驗室手冊中。Retrovirus vectors and adeno-associated viral vectors can be used as recombinant gene delivery systems for the transfer of foreign nucleic acid molecules encoding fusion proteins in vivo. Such vectors efficiently deliver nucleic acids into cells and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of a specialized cell line (called a "set of cells") that only produces a replication-defective retrovirus has increased the availability of retroviruses in gene therapy, and the defective retroviruses have been characterized by characterization. For gene transfer for gene therapy purposes (for a review of this method, see Miller, AD (1990) Blood 76: 271). The replication-defective virus can be assembled into virions that can be labeled by standard techniques and using helper viruses. Procedures for generating recombinant retroviruses and protocols for infecting cells with such viruses in vitro or in vivo can be documented in existing protocols in molecular biology (Current Protocols in Molecular Biology, Ausubel, FM et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14) and other standard laboratory manuals.

另一有用的病毒基因輸送系統係使用衍生自腺病毒之載體。可將腺病毒之基因組加以處理，以致其編碼及表現所探討之基因產物，但在正常溶裂病毒生命循環中之複製能力則喪失。例如參見BerKner et al.,BioTechniques 6：616(1988)；Rosenfeld et al.,Science 252：431-434(1991)；以及Rosenfeld et al.,Cell 68：143-155(1992)。衍生自腺病毒Ad株5型d1324或腺病毒之其他株之適當腺病毒載體(例如Ad2,Ad3,Ad7等)為精於本技術者所已知。重組腺病毒在某些情況下為有利，因其不會感染未***細胞且可被用於感染廣泛種類之細胞類型，包括上皮細胞(Rosenfeld et al.,(1992)，如上文所引用者)。再者，該病毒粒子相對安定及可進行純化及濃縮，且如上述可加以修改，以影響感染力之範圍。此外，引進之腺病毒DNA(以及其所含之外來DNA)未被整合入宿主細胞之基因組而保持為游離基因體(episomal)，藉此可避免引進之DNA整合入宿主基因組(例如反錄DNA)時於整合位置發生***性突變所引起之潛在問題。再者，腺病毒基因組攜帶外來DNA之能力比其他基因輸送用載體大(高達8仟鹼基)(Berkner et al.,上文所引用者；Haj-Ahmand et al.,J.Virol.57：267(1986))。Another useful viral gene delivery system uses a vector derived from an adenovirus. The genome of the adenovirus can be processed such that it encodes and expresses the gene product under investigation, but the ability to replicate in the normal lytic virus life cycle is lost. See, for example, BerKner et al., BioTechniques 6: 616 (1988); Rosenfeld et al., Science 252: 431-434 (1991); and Rosenfeld et al., Cell 68: 143-155 (1992). Suitable adenoviral vectors (e.g., Ad2, Ad3, Ad7, etc.) derived from adenovirus Ad strain type 5 d1324 or other strains of adenovirus are known to those skilled in the art. Recombinant adenoviruses are advantageous in some cases because they do not infect undivided cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992), cited above) . Furthermore, the virions are relatively stable and can be purified and concentrated, and can be modified as described above to affect the range of infectivity. In addition, the introduced adenoviral DNA (and the foreign DNA it contains) is not integrated into the genome of the host cell and remains as episomal, thereby preventing the introduction of the introduced DNA into the host genome (eg, re-recorded DNA) The potential problem caused by insertional mutations at the integration site. Furthermore, the adenoviral genome is capable of carrying foreign DNA larger than other gene delivery vectors (up to 8 bases) (Berkner et al., cited above; Haj-Ahmand et al., J. Virol. 57: 267 (1986)).

在另一具體例中，本發明之非病毒基因輸送系統依賴細胞吞噬途徑(endocytic pathways)而使標定細胞攝入目標核苷酸分子。例示之此型基因輸送系統包括衍生自微脂粒之系統、聚離胺酸綴合物及人造病毒套膜。在代表性具體例中，編碼本發明之融合蛋白質之核酸分子可封入表面帶正電之微脂粒[例如親脂質(lipofectins)]中且(視需要)核酸分子可用對抗目標組織之細胞表面抗原之抗體加以標記(Mizuno et al.(1992)No Shinkei Geka 20：547-551；PCT公開WO91/06309；日本專利申請案第1047381號；以及歐洲專利公開EP-A43075)。In another embodiment, the non-viral gene delivery system of the invention relies on endocytic pathways to allow the labeled cells to uptake the target nucleotide molecule. Exemplary such gene delivery systems include systems derived from vesicles, polylysine conjugates, and artificial viral envelopes. In a representative embodiment, the nucleic acid molecule encoding the fusion protein of the present invention can be encapsulated in a surface positively charged liposome [e.g., lipofectins] and (if desired) a nucleic acid molecule can be used against the cell surface antigen of the target tissue. The antibody is labeled (Mizuno et al. (1992) No Shinkei Geka 20: 547-551; PCT Publication WO 91/06309; Japanese Patent Application No. 1043781; and European Patent Publication EP-A43075).

編碼本發明融合蛋白質之基因所用之基因輸送系統可藉由許多方法之任一者引進病患中。舉例言之，基因輸送系統之醫藥製劑可全身性地，例如藉由靜脈注射引進，而在目標細胞中蛋白質之特異性轉導主要係基於基因輸送載體所提供之特異性轉染、由轉錄調節序列控制之受體基因表現所造成之細胞型或組織型表現、或其組合。在其他具體例中，重組基因之初始輸送較為侷限，在動物中之引進十分局部化。舉例言之，基因輸送載體可經由導管(參見美國專利第5,328,470號)或經由立體定向注射(例如Chen et al.(1994)PNAS 91：3054-3057)引進。基因治療構築體之醫藥製劑可主要由在可接受稀釋劑中之基因輸送系統組成，或者可包含包埋有基因輸送載劑之緩釋基質。在融合蛋白質可由重組細胞例如反錄病毒載體完整產生之情況，醫藥組合物可包含一種或一種以上會產生融合蛋白質之細胞。A gene delivery system for use in encoding a gene for a fusion protein of the present invention can be introduced into a patient by any of a number of methods. For example, the pharmaceutical preparation of the gene delivery system can be introduced systemically, for example by intravenous injection, and the specific transduction of the protein in the target cell is mainly based on the specific transfection provided by the gene delivery vector, regulated by transcription. The cell type or tissue type manifestation resulting from the expression of the sequence-controlled receptor gene, or a combination thereof. In other specific examples, the initial delivery of recombinant genes is more limited and the introduction in animals is very localized. For example, the gene delivery vector can be introduced via a catheter (see U.S. Patent No. 5,328,470) or via stereotactic injection (e.g., Chen et al. (1994) PNAS 91: 3054-3057). The pharmaceutical preparation of the gene therapy construct may consist essentially of a gene delivery system in an acceptable diluent, or may comprise a sustained release matrix entrapped with a gene delivery vehicle. Where the fusion protein is fully produced by a recombinant cell, such as a retroviral vector, the pharmaceutical composition may comprise one or more cells that will produce the fusion protein.

在另一具體例中，使用RNA干擾(RNAi)降低或完全抑制先天性免疫受體之表現，其中該先天性免疫受體依照本文揭示之方法被鑑定為參與致病過程。RNAi為本技術所熟知且可藉由使用小型干擾性RNA(siRNA)達成。依照本發明之siRNAs具有達29 bps、25 bps、22 bps、21 bps、20 bps、15 bps、10 bps、5 bps或在上述數值附近或之間之任何整數。該等siRNAs可以例如載體(例如編碼小型髮夾狀RNA(shRNA)之載體)所編碼之形式或以微脂粒核酸複合物投與。脂質：核酸複合物(包括標定之微脂粒諸如免疫脂質複合物)之製備為精於本技術者所熟知(例如參見Crystal,Science 270：404-410(1995)；Blaese et al.,Cancer Gene Ther.2：291-297(1995)；Behr et al.,Bioconjugate Chem,5：382-389(1994)；Remy et al.,Bioconjugate Chem.5：647-654(1994)；Gao et al.,Gene Therapy 2：710-722(1995)；Ahmad et al.,Cancer Res.52：4817-4820(1992)；美國專利第4,186,183號、第4,217,344號、第4,235,871號、第4,261,975號、第4,485,054號、第4,501,728號、第4,774,085號、第4,837,028號及第4,946,787號)。所以，本發明亦提供包含RNA分子之醫藥組合物，該RNA當投與至病患時，能媒介先天性免疫受體之RNA干擾。In another embodiment, RNA interference (RNAi) is used to reduce or completely inhibit the performance of an innate immune receptor, wherein the innate immune receptor is identified as participating in a pathogenic process in accordance with the methods disclosed herein. RNAi is well known in the art and can be achieved by the use of small interfering RNA (siRNA). The siRNAs according to the invention have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integer near or between the above values. Such siRNAs can be administered, for example, in the form encoded by a vector (e.g., a vector encoding a small hairpin RNA (shRNA)) or in a liposome nucleic acid complex. The preparation of lipid:nucleic acid complexes, including calibrated liposomes such as immunolipid complexes, is well known to those skilled in the art (see, for example, Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene). Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem, 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2: 710-722 (1995); Ahmad et al., Cancer Res. 52: 4817-4820 (1992); U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, Nos. 4,501,728, 4,774,085, 4,837,028 and 4,946,787). Therefore, the present invention also provides a pharmaceutical composition comprising an RNA molecule which, when administered to a patient, is capable of mediating RNA interference of an innate immune receptor.

根據具體例，本文提供在巨噬細胞中由RNAi所媒介之DVLR1/CLEC5A基因表現下降(knock down)的非限定性實例。以此方式產生之DVLR1/CLEC5A減活(attenuation)顯著降低感染登革熱病毒(DV)之巨噬細胞中促發炎性細胞激素之分泌，因而表示RNAi所媒介之DVLR1/CLEC5A減活在治療登革熱病毒上有用。Illustrative examples provide non-limiting examples of the knockdown of the DVLR1/CLEC5A gene mediated by RNAi in macrophages. The DVLR1/CLEC5A attenuation produced in this way significantly reduces the secretion of pro-inflammatory cytokines in macrophages infected with dengue virus (DV), thus indicating that RNAi-mediated DVLR1/CLEC5A is deactivated in the treatment of dengue virus it works.

減少DVLR1/CLEC5A表現之siRNA或shRNA被特別考量用於登革熱患者之RNAi-媒介治療。設計、合成及投與shRNA及siRNA以減少特異性基因表現之方法為本技術所熟知且記載於，例如，美國專利第7,022,828號。適於與本發明之shRNA構築體及siRNA分子一起調配之藥劑之非限定性例子包括：與PEG綴合之核酸、與磷脂綴合之核酸、含有親脂部分之核酸、硫代磷酸酯(phosphorothioates)、P-醣蛋白抑制劑(諸如Pluronic P85)[其可促進藥物進入各種組織，例如CNS(Jolliet-Riant and Tillement,1999,Fundam.Clin.Pharmacol.,13,1626)]；生物可降解聚合物，諸如植入後持續釋放輸送用之聚(DL-丙交酯-乙交酯)微球囊(Emerich,DF et al,1999,Cell Transplant,8,47 58)Alkermes,Inc.Cambridge,Mass.；以及有負載之奈米粒子諸如由聚氰基丙烯酸丁酯製成者，其可將藥物輸送通過血腦障壁且可改變神經元攝取機制(Prog Neuropsychopharmacol Biol Psychiatry,23,941 949,1999)。輸送手段(包括本發明之核酸分子之CNS輸送)之其他非限定性例子包括下列文獻所述之材料：Boado et al.,1998,J.Pharm.Sci.,87,1308 1315；Tyler et al,1999,FEBS Lett.,421,280 284；Pardridge et al.,1995,PNAS USA.,92,5592 5596；Boado,1995,Adv.Drug Delivery Rev.,15,73 107；Aldrian-Herrada et al.,1998,Nucleic Acids Res.,26,4910 4916；及Tyler et al.,1999,PNAS USA.,96,7053 7058。所有此等文獻以參考資料方式納入本文。此外，包含表面經修改之微脂粒之組合物，其中該表面經修改之微脂粒含有聚(乙二醇)脂質，即經PEG修改或長時間循環之微脂粒或長命微脂粒(stealth liposomes)，亦可與本發明之核酸合用。本發明之核酸分子亦包含共價連接之具有各種分子量之PEG分子。此等調配物提供在目標組織中增加藥物蓄積之方法。該類藥物載劑可阻止藥物被單核吞食系統(MPS或RES)調理(opsonization)及清除，藉此使得經包納之藥物在血液循環中之時間增長及組織暴露增加(Lasic et al.Chem.Rev.1995,95,2601 2627；Ishiwata et al.,Chem.Pharm.Bull.1995,43,1005 1011)。已證明該等微脂粒會選擇性地蓄積在腫瘤中，此可能係經由在血管新生化目標組織中之血管外滲及擷取而產生(Lasic et al.,Science 1995,267,1275 1276；Oku et al.,1995,Biochim.Biophys.Acta,1238,86 90)。長時間循環微脂粒，尤其當與已知會蓄積在MPS組織中之習知陽離子性微脂粒相較時，會促進DNA及RNA之藥品動力及藥品靜力性質(Liu et al.,J.Biol.Chem.1995,42,24864 24870；Choi et al.,國際PCT公開號WO 96/10391；Ansell et al.,國際PCT公開號WO 96/10390；Holland et al.,國際PCT公開號WO 96/10392；所有此等文獻皆以參考資料方式納入本文)。長時間循環微脂粒由於能避免蓄積在代謝旺盛之單核吞食系統(MPS)組織諸如肝及脾，因此與陽離子性微脂粒相較，對於藥物可能有較大的保護作用，使其免於被核酸酶降解。siRNA or shRNA that reduces DVLR1/CLEC5A expression is specifically considered for RNAi-mediated therapy in dengue patients. Methods for designing, synthesizing, and administering shRNAs and siRNAs to reduce specific gene expression are well known in the art and are described, for example, in U.S. Patent No. 7,022,828. Non-limiting examples of agents suitable for formulation with the shRNA constructs and siRNA molecules of the invention include: nucleic acids conjugated to PEG, nucleic acids conjugated to phospholipids, nucleic acids containing lipophilic moieties, phosphorothioates a P-glycoprotein inhibitor (such as Pluronic P85) [which promotes drug entry into various tissues, such as the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 1626)]; biodegradable polymerization , such as poly (DL-lactide-glycolide) microspheres for sustained release after delivery (Emerich, DF et al, 1999, Cell Transplant, 8, 47 58) Alkermes, Inc. Cambridge, Mass And loaded nanoparticles such as those made of polybutyl cyanoacrylate, which deliver drugs through the blood brain barrier and alter the neuronal uptake mechanism (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941 949, 1999). Other non-limiting examples of delivery means, including CNS delivery of nucleic acid molecules of the invention, include those described in Boado et al., 1998, J. Pharm. Sci., 87, 1308 1315; Tyler et al, 1999, FEBS Lett., 421, 280 284; Pardridge et al., 1995, PNAS USA., 92, 5592 5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73 107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910 4916; and Tyler et al., 1999, PNAS USA., 96, 7053 7058. All such documents are incorporated herein by reference. Further, a composition comprising a surface modified aliquot, wherein the modified microlipid contains poly(ethylene glycol) lipids, ie, PEG modified or long-circulating aliquots or long-lived vesicles ( The stealth liposomes) can also be used in combination with the nucleic acids of the invention. The nucleic acid molecules of the invention also comprise covalently linked PEG molecules of various molecular weights. Such formulations provide a means of increasing drug accumulation in the target tissue. This drug carrier prevents the drug from being opsonized and cleared by the mononuclear system (MPS or RES), thereby increasing the time in the blood circulation and increasing tissue exposure (Lasic et al.Chem) .Rev. 1995, 95, 2601 2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005 1011). These vesicles have been shown to selectively accumulate in tumors, possibly through extravascular osmosis and extraction in neovascularized target tissues of blood vessels (Lasic et al., Science 1995, 267, 1275 1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86 90). Long-term circulation of liposomes, especially when compared to conventional cationic vesicles known to accumulate in MPS tissue, promotes drug and drug static properties of DNA and RNA (Liu et al., J. Biol. Chem. 1995, 42, 24864 24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96 /10392; all such references are incorporated herein by reference. Long-term circulating microlipids can avoid accumulation in the metabolically active mononuclear ingestion system (MPS) tissues such as liver and spleen. Therefore, compared with cationic vesicles, it may have a greater protective effect on drugs. Degraded by nucleases.

實施例Example

本發明藉由下列非限定性實施例進一步說明：The invention is further illustrated by the following non-limiting examples:

實施例1：先天性免疫受體：Fc融合蛋白質之製備Example 1: Innate immune receptor: preparation of Fc fusion protein 細胞培養物Cell culture

將293F細胞(Invitrogen,R790-07)在置於回轉式振盪器(125rpm)上之125mL燒瓶內，於不含血清之293FREESTYLE 203表現培養基(Invitrogen,12338-018)中，於37℃在CO₂ 培育器內培養。293F cells (Invitrogen, R790-07) were placed in a 125 mL flask placed on a rotary shaker (125 rpm) in serum-free 293FREESTYLE 203 expression medium (Invitrogen, 12338-018) at 37 ° C in CO ₂ Culture in the incubator.

受體.Fc融合基因之構築Construction of receptor.Fc fusion gene

凝集素受體之細胞外區域，TREMs及TLTs，藉由反錄酶聚合酶連鎖反應(RT-PCR)選殖，繼而次選殖入yT&A載體，然後選殖入pcDNA3.1(+)hIgG1.Fc表現載體。生成之受體.Fc構築體編碼與經突變之人類IgG1 Fc部分(其不會與人類Fc受體結合)融合之重組蛋白質。在IgG1 Fc部分中之突變為L234A、L235E、G237A及P331S。用於以RT-PCR擴增細胞外區域之引子之序列如下(或者可自表2所列之序列選擇引子)：The extracellular domain of lectin receptor, TREMs and TLTs, were cloned by reverse transcription enzyme polymerase chain reaction (RT-PCR), then subcultured into yT&A vector, and then cloned into pcDNA3.1(+)hIgG1. Fc expression vector. The resulting receptor.Fc construct encodes a recombinant protein fused to a mutated human IgGl Fc portion that does not bind to a human Fc receptor. The mutations in the IgG1 Fc portion were L234A, L235E, G237A and P331S. The sequences of the primers used to amplify the extracellular region by RT-PCR are as follows (or the primers can be selected from the sequences listed in Table 2):

CLEClA/CLEC-1CLEClA/CLEC-1

意義引子Meaning primer

5’-GAATCCTTTCAGTACTACCAGCTCTCC-3’5’-GAATCCTTTCAGTACTACCAGCTCTCC-3’

序列編號：1Serial number: 1

反義引子Antisense primer

5’-GAATTCTCAGTCACCTTCGCCTAATGT-3’5’-GAATTCTCAGTCACCTTCGCCTAATGT-3’

序列編號：2Serial number: 2

CLEC1B/CLEC-2CLEC1B/CLEC-2

意義引子Meaning primer

5’-GGATCCCTGGGGATTTGGTCTGTC-3’5’-GGATCCCTGGGGATTTGGTCTGTC-3’

序列編號：3Serial number: 3

反義引子Antisense primer

5’-GAATTCTTAAGGTAGTTGGTCCAC-3’5’-GAATTCTTAAGGTAGTTGGTCCAC-3’

序列編號：4Serial number: 4

CLEC2B/AICLCLEC2B/AICL

意義引子Meaning primer

5'-GGATCCTCTCAGAGTTTATGCCCC-3'5'-GGATCCTCTCAGAGTTTATGCCCC-3'

序列編號：5Serial number: 5

反義引子Antisense primer

5'-GGATCCCCCCATTATCTTAGACAT-3'5'-GGATCCCCCCATTATCTTAGACAT-3'

序列編號：6Serial number: 6

CLEC4A/DCIRCLEC4A/DCIR

意義引子Meaning primer

5’-GGATCCTTTCAAAAATATTCTCAGCTTCTT-3’5’-GGATCCTTTCAAAAATATTCTCAGCTTCTT-3’

序列編號：7Serial number: 7

反義引子Antisense primer

5’-GAATTCTCATAAGTGGATCTTCATCATC-3’5’-GAATTCTCATAAGTGGATCTTCATCATC-3’

序列編號：8Serial number: 8

CLEC4C/BDCA-2CLEC4C/BDCA-2

意義引子Meaning primer

5’-GGATCCTTTATGTATAGCAAAACTGTCAAG-3’5’-GGATCCTTTATGTATAGCAAAACTGTCAAG-3’

序列編號：9Serial number: 9

反義引子Antisense primer

5’-GAATTCTTATATGTAGATCTTCTTCATCTT-3’5’-GAATTCTTATATGTAGATCTTCTTCATCTT-3’

序列編號：10Serial number: 10

CLEC4D/CLEC-6CLEC4D/CLEC-6

意義引子Meaning primer

5’-GAATCCCATCACAACTTTTCACGCTGT-3’5’-GAATCCCATCACAACTTTTCACGCTGT-3’

序列編號：11Serial number: 11

反義引子Antisense primer

5’-GAATTCCTAGTTCAATGTTGTTCCAGG-3’5’-GAATTCCTAGTTCAATGTTGTTCCAGG-3’

序列編號：12Serial number: 12

CLEC4E/MINCLECLEC4E/MINCLE

意義引子Meaning primer

5’-GAAGATCTACATTTCGCATCTTTCAAACC-3’5’-GAAGATCTACATTTCGCATCTTTCAAACC-3’

序列編號：13Serial number: 13

反義引子Antisense primer

5’-GCGGTTAAAGAGATTTTCCTTTGTTCA-3’5’-GCGGTTAAAGAGATTTTCCTTTGTTCA-3’

序列編號：14Serial number: 14

CLEC4K/郎罕細胞特異蛋白CLEC4K/ Langhan cell specific protein

意義引子Meaning primer

5’-GGATCCCGGTTTATGGGCACCATA-3’5’-GGATCCCGGTTTATGGGCACCATA-3’

序列編號：15Serial number: 15

反義引子Antisense primer

5’-GGATCCTCACGGTTCTGATGGGAC-3’5’-GGATCCTCACGGTTCTGATGGGAC-3’

序列編號：16Serial number: 16

CLEC4L/DC-SIGNCLEC4L/DC-SIGN

意義引子Meaning primer

5’-GGATCCAAGGTCCCCAGCTCCATAAG-3’5’-GGATCCAAGGTCCCCAGCTCCATAAG-3’

序列編號：17Serial number: 17

反義引子Antisense primer

5’-GAATTCCTACGCAGGAGGGGGGT-3’5’-GAATTCCTACGCAGGAGGGGGGT-3’

序列編號：18Serial number: 18

CLEC4M/DC-SIGNR/L-SIGNCLEC4M/DC-SIGNR/L-SIGN

意義引子Meaning primer

5’-GGATCCTCCAAGGTCCCCAGCTCC-3’5’-GGATCCTCCAAGGTCCCCAGCTCC-3’

序列編號：19Serial number: 19

反義引子Antisense primer

5’-GAATTCCTATTCGTCTCTGAAGCAGG-3’5’-GAATTCCTATTCGTCTCTGAAGCAGG-3’

序列編號：20Serial number: 20

DTVR1/CTFC5A(MDL-l)DTVR1/CTFC5A (MDL-l)

意義引子Meaning primer

5’-AGATCTAGTAACGATGGTTTCACCAC-3’5’-AGATCTAGTAACGATGGTTTCACCAC-3’

序列編號：21Serial number: 21

反義引子Antisense primer

5’-GAATTCCTGTGATCATTTGGCATTCTT-3’5’-GAATTCCTGTGATCATTTGGCATTCTT-3’

序列編號：22Serial number: 22

CLEC6A/樹狀細胞凝集素-2CLEC6A/dendritic cell agglutinin-2

意義引子Meaning primer

5’-GGATCCACATATGGTGAAACTGGC-3’5’-GGATCCACATATGGTGAAACTGGC-3’

序列編號：23Serial number: 23

反義引子Antisense primer

5’-GAATTCCATCAGTCGATGGGC-3’5’-GAATTCCATCAGTCGATGGGC-3’

序列編號：24Serial number: 24

CLEC7A/樹狀細胞凝集素-1CLEC7A/dendritic cell lectin-1

意義引子Meaning primer

5’-GGATCCACCATGGCTATTTGGAGATCC-3’5’-GGATCCACCATGGCTATTTGGAGATCC-3’

序列編號：25Serial number: 25

反義引子Antisense primer

5’-GAATTCTTACATTGAAAACTTCTTCTCACA-3’5’-GAATTCTTACATTGAAAACTTCTTCTCACA-3’

序列編號：26Serial number: 26

CLEC10A/ML2CLEC10A/ML2

意義引子Meaning primer

5’-GGATCCTCCAAATTTCAGAGGGACCTG-3’5’-GGATCCTCCAAATTTCAGAGGGACCTG-3’

序列編號：27Serial number: 27

反義引子Antisense primer

5’-GAATTCTCAGTGACTCTCCTGGCTG-3’5’-GAATTCTCAGTGACTCTCCTGGCTG-3’

序列編號：28Serial number: 28

CLEC12A/CLL-1CLEC12A/CLL-1

意義引子Meaning primer

5’-GGATCCGTAACTTTGAAGATAGAAATGAAA-3’5’-GGATCCGTAACTTTGAAGATAGAAATGAAA-3’

序列編號：29Serial number: 29

反義引子Antisense primer

5’-GAATCCTCATGCCTCCCTAAAATATGTA-3’5’-GAATCCTCATGCCTCCCTAAAATATGTA-3’

序列編號：30Serial number: 30

CLEC13A/BIMLECCLEC13A/BIMLEC

意義引子Meaning primer

5’-GGATCCTCATGCTCCGGGCCGCG-3’5’-GGATCCTCATGCTCCGGGCCGCG-3’

序列編號：31Serial number: 31

反義引子Antisense primer

5’-GAATTCGCTAGCAATCACCAATGCTGA-3’5’-GAATTCGCTAGCAATCACCAATGCTGA-3’

序列編號：32Serial number: 32

COLEC12/CL-P1COLEC12/CL-P1

意義引子Meaning primer

5’-AGAGGTGACAGAGGATCCCA-3’5’-AGAGGTGACAGAGGATCCCA-3’

序列編號：33Serial number: 33

反義引子Antisense primer

5’-GAATTCGTGATCCCATCACAGTCC-3’5’-GAATTCGTGATCCCATCACAGTCC-3’

序列編號：34Serial number: 34

MAFA-L/KLRG-1MAFA-L/KLRG-1

意義引子Meaning primer

5’-GGATCCTGCCAGGGCTCCAACT-3’5’-GGATCCTGCCAGGGCTCCAACT-3’

序列編號：35Serial number: 35

反義引子Antisense primer

5’-ATGACAGATCTGAGGGTCA-3’5’-ATGACAGATCTGAGGGTCA-3’

序列編號：36Serial number: 36

重組受體.Fc融合蛋白質之表現及純化Expression and purification of recombinant receptor.Fc fusion protein

受體Fc蛋白質藉由使用FREESTYLE 293表現系統(Invitrogen,Carlsbad,CA)過度表現並在蛋白質A管柱上純化。簡言之，將3 X 10⁷ 293-F細胞以1,500rpm旋轉沈澱，繼而重新懸浮於28 ml FREESTYLE 293表現培養基中。然後將40μl之293FECTIN與1 ml OPTI-MEM(Invitrogen,31985-062)於室溫混合5分鐘，然後與30 μg質體DNA在1 ml OPTI-MEM(Invitrogen,31985-062)中再培育20分鐘後，加至293-F細胞中。48小時後，收取上清液並藉由蛋白質A管柱純化重組融合蛋白質。The receptor Fc protein was overexpressed by using the FREESTYLE 293 Expression System (Invitrogen, Carlsbad, CA) and purified on a Protein A column. Briefly, 3 X 10 ⁷ 293-F cells were spun down at 1,500 rpm and then resuspended in 28 ml FREESTYLE 293 expression medium. Then, 40 μl of 293FECTIN was mixed with 1 ml of OPTI-MEM (Invitrogen, 31985-062) for 5 minutes at room temperature, and then incubated with 30 μg of plastid DNA in 1 ml of OPTI-MEM (Invitrogen, 31985-062) for 20 minutes. After that, it was added to 293-F cells. After 48 hours, the supernatant was collected and the recombinant fusion protein was purified by Protein A column.

實施例2：多醣體萃取物之製備Example 2: Preparation of polysaccharide extract 靈芝之粗製萃取物Ganoderma lucidum crude extract

粗製靈芝萃取物(經由鹼性萃取，中和及乙醇沉澱而製備)得自Pharmanex公司(CA,USA)。除非另有指示，分子量排除界線(MWCO)為6000-8000道爾頓之Spectrapor管狀透析膜、Thermo bio-basic SEC-1000管柱、Tosoh TSK G5000PWx1 SEC管柱，以及所有化學品及試劑得自Sigma或Aldrich公司。Crude Ganoderma lucidum extract (prepared via alkaline extraction, neutralization and ethanol precipitation) was obtained from Pharmanex Corporation (CA, USA). Spectrapor with a molecular weight exclusion boundary (MWCO) of 6000-8000 Daltons unless otherwise indicated Tubular dialysis membranes, Thermo bio-basic SEC-1000 tubing, Tosoh TSK G5000 PWx1 SEC tubing, and all chemicals and reagents were obtained from Sigma or Aldrich.

靈芝萃取物之純化Purification of Ganoderma Lucidum Extract

將粗製靈芝粉末(6g)(得自Pharmanex公司)溶於120ml之ddH₂ O，於沸水(100℃)中攪拌2小時並離心(1000 rpm)1小時，以移除不溶的物質。將生成之溶液在約40℃與約50℃之間濃縮以得到小體積，然後冷凍乾燥，產生5g(83%)暗褐色之粉末(靈芝多醣體；GLPS)。將該水溶性殘餘物貯存於-20℃直至進一步純化。Crude Ganoderma lucidum powder (6 g) (available from Pharmanex) was dissolved in 120 ml of ddH ₂ O, stirred in boiling water (100 ° C) for 2 hours and centrifuged (1000 rpm) for 1 hour to remove insoluble matter. The resulting solution was concentrated between about 40 ° C and about 50 ° C to give a small volume, which was then lyophilized to give 5 g (83%) of dark brown powder (Ganoderma lucidum polysaccharide; GLPS). The water soluble residue was stored at -20 °C until further purification.

靈芝多醣體之F3部分之標準化-單離Standardization of the F3 part of Ganoderma lucidum polysaccharides

自靈芝多醣體之水溶性殘餘物之暗色粉末單離靈芝多醣體部分3(下文稱為“GLPS F3”及“F3”)。所有層析步驟在冷凍室中於4℃進行。將樣品(2.1g)溶於小體積之含0.1N疊氮化鈉之Tris緩衝液(pH 7.0,0.1 N)中，並藉由凝膠過濾層析純化，其中使用Sephacryl S-500管柱(95 X 2.6 cm)及以0.1 N Tris緩衝液(pH 7.0)做為溶析劑。將流速設定為0.6 mL/分鐘並每管收集6.0 mL。層析後，各部分以酚-H₂ SO₄ 法檢測各試管中之糖含量。收集5部分(部分1-5)。將部份3(F3)在旋轉蒸發器中於約40至50℃濃縮以得到小體積，然後將其用6000-8000道爾頓MWCO膜進行透析，以移除過多的鹽及疊氮化鈉。透析後，將F3冷凍乾燥，得到520 mg固體。The dark powder of the water-soluble residue of the Ganoderma lucidum polysaccharide is separated from the Ganoderma lucidum polysaccharide fraction 3 (hereinafter referred to as "GLPS F3" and "F3"). All chromatography steps were carried out in a freezer at 4 °C. The sample (2.1 g) was dissolved in a small volume of 0.1 N sodium azide in Tris buffer (pH 7.0, 0.1 N) and purified by gel filtration chromatography using a Sephacryl S-500 column ( 95 X 2.6 cm) and 0.1 N Tris buffer (pH 7.0) as a solvent. The flow rate was set to 0.6 mL/min and 6.0 mL was collected per tube. After chromatography, the fractions were tested for sugar content in each tube by the phenol-H ₂ SO ₄ method. Collect 5 parts (parts 1-5). Part 3 (F3) was concentrated in a rotary evaporator at about 40 to 50 ° C to give a small volume, which was then dialyzed against a 6000-8000 Dalton MWCO membrane to remove excess salt and sodium azide. . After dialysis, F3 was lyophilized to give 520 mg of a solid.

來自冬蟲夏草之多醣體之製備Preparation of polysaccharides from Cordyceps sinensis

為了純化來自冬蟲夏草之多醣體，將樣品切成0.2 cm³ 片，在去離子化沸水(100℃)中培育60分鐘，然後冷卻至室溫，接著通過0.2μm濾器，繼而藉由加入等體積之乙醇以沉澱出多醣體。將沉澱物用冷凍乾燥器乾燥並貯存於4℃。多醣體之總糖分析藉由酚-H₂ SO₄ 法，測量485 nm之OD而測定；而多醣體之純度藉由使用Thermo Bio-Basic SEC-1000管柱進行HPLC而測定，其中使用RI檢測器於280 nm進行UV檢測。To purify the polysaccharide from Cordyceps sinensis, the sample was cut into 0.2 cm ³ pieces, incubated in deionized boiling water (100 ° C) for 60 minutes, then cooled to room temperature, then passed through a 0.2 μm filter, followed by the addition of an equal volume. Ethanol precipitates the polysaccharide. The precipitate was dried in a freeze dryer and stored at 4 °C. The total sugar analysis of the polysaccharide was determined by measuring the OD at 485 nm by the phenol-H ₂ SO ₄ method; and the purity of the polysaccharide was determined by HPLC using a Thermo Bio-Basic SEC-1000 column using RI detection. UV detection was performed at 280 nm.

來自霍山石斛之多醣體之製備Preparation of polysaccharides from Dendrobium huoshanense

將風乾之霍山石斛壓碎、磨成粉末、在蒸餾水中均質化並於4℃攪拌整夜。不溶之物質藉由離心收集。將上清液濃縮成小體積，然後加入1體積乙醇，以得到沉澱物(O)及上清液(N)。將TSK G-5000 PW尺寸排除管柱用於高效液相層析以進行多醣體分析，並使用具有界定分子量之標準普魯籣多醣(pullulan)部分。據評估，上清液(N)中之多醣體之分子量在1.2x10⁵ 至4.1x10⁵ 道爾頓之間，而沉澱物(O)中多醣體之分子量在1.0x10⁶ 至2.2x10⁵ 道爾頓之間。碳水化合物總含量藉由酚-硫酸法測量。在沉澱物(O)中之多醣體為83%，而在上清液(N)中之多醣體為77%。沉澱物(O)及上清液(N)二者之碘反應試驗皆為陽性(λmax 440 nm，深藍色)，此暗示在此等部分中之多醣體主要為α-D-葡聚糖。The air-dried Huoshan Dendrobium was crushed, ground into a powder, homogenized in distilled water and stirred overnight at 4 °C. Insoluble materials are collected by centrifugation. The supernatant was concentrated to a small volume, and then 1 volume of ethanol was added to obtain a precipitate (O) and a supernatant (N). The TSK G-5000 PW size exclusion column was used for high performance liquid chromatography for polysaccharide analysis and a standard pullulan fraction with defined molecular weight was used. According to the evaluation, the supernatant (N), the molecular weight of the polysaccharide of between 1.2x10 ⁵ to 4.1x10 ⁵ Daltons, and the molecular weight of the precipitate (O) in the polysaccharides of 1.0x10 ⁶ to 2.2x10 ⁵ Doyle Between the two. The total carbohydrate content is measured by the phenol-sulfuric acid method. The polysaccharide in the precipitate (O) was 83%, and the polysaccharide in the supernatant (N) was 77%. Both the precipitate (O) and the supernatant (N) were positive for the iodine reaction (λmax 440 nm, dark blue), suggesting that the polysaccharides in these fractions are predominantly alpha-D-glucans.

來自蘑菇之多醣體之製備Preparation of polysaccharides from mushrooms

將風乾之蘑菇(Lentinus edodes)壓碎、磨成粉末、在蒸餾水中均質化及於4℃攪拌整夜。殘餘物藉由離心移除並將上清液濃縮成小體積，然後冷凍乾燥，得到粗製之多醣體L。之後，將0.25N NaOH溶液加至不溶於水之殘餘物中(該殘餘物藉由離心單離)，將該混合物於室溫攪拌整夜，之後加入2體積之乙醇以沉澱出多醣體。然後將蒸餾水加至沉澱出之多醣體中，接著加入乙酸中和pH值。將生成之溶液離心及冷凍乾燥，得到多醣體M。然後使用TSK G-5000 PW尺寸排除管柱進行HPLC，以分析多醣體。碳水化合物總含量藉由酚-硫酸法測量，結果L包含79%碳水化合物且M包含90%碳水化合物。與來自蘑菇之多醣體部分之數據之比較暗示多醣體L及M主要為β-1,3-D-葡聚糖。The air-dried mushrooms (Lentinus edodes) were crushed, ground to a powder, homogenized in distilled water and stirred overnight at 4 °C. The residue was removed by centrifugation and the supernatant was concentrated to a small volume, which was then lyophilized to give a crude polysaccharide L. Thereafter, a 0.25 N NaOH solution was added to the water-insoluble residue (the residue was isolated by centrifugation), and the mixture was stirred at room temperature overnight, after which 2 volumes of ethanol were added to precipitate a polysaccharide. Distilled water was then added to the precipitated polysaccharide, followed by the addition of acetic acid to neutralize the pH. The resulting solution was centrifuged and freeze-dried to obtain a polysaccharide M. The TSK G-5000 PW size exclusion column was then used for HPLC to analyze the polysaccharide. The total carbohydrate content was measured by the phenol-sulfuric acid method, and as a result, L contained 79% of carbohydrates and M contained 90% of carbohydrates. Comparison with the data from the polysaccharide fraction of the mushroom suggests that the polysaccharides L and M are mainly β-1,3-D-glucan.

β-1,3-葡聚糖、D-葡萄糖及D-半乳糖之製備Preparation of β-1,3-glucan, D-glucose and D-galactose

為了製備競爭性檢定分析用之樣品，將100 mg之β-1,3-葡聚糖(Fluka,Japan)懸浮於7.5 ml水中，並加入50 μl之40%(w/w)氫氧化鈉水溶液。將該混合物回流加熱1.5小時並冷卻。然後加入甲醇以沉澱出β-1,3-葡聚糖。將β-1,3-葡聚糖沉澱物溶於水，對4 L dd-H₂ O透析4次，並於減壓下濃縮，得到水溶性β-1,3-葡聚糖。將D-葡萄糖(Sigma)及D-半乳糖(Sigma)溶於dd-H₂ O(100 mg/ml)並貯存於4℃。To prepare a sample for competitive assay analysis, 100 mg of β-1,3-glucan (Fluka, Japan) was suspended in 7.5 ml of water and 50 μl of 40% (w/w) aqueous sodium hydroxide solution was added. . The mixture was heated under reflux for 1.5 hours and cooled. Methanol was then added to precipitate β-1,3-glucan. The β-1,3-glucan precipitate was dissolved in water, dialyzed against 4 L of dd-H ₂ O 4 times, and concentrated under reduced pressure to give water-soluble β-1,3-glucan. D-glucose (Sigma) and D-galactose (Sigma) were dissolved in dd-H ₂ O (100 mg/ml) and stored at 4 °C.

生物素基-F3之製備Preparation of biotinyl-F3

將靈芝多醣體-F3使用生物素以「單槽」(onepot)反應標記。特定而言，使在0.2 N NaHCO₃ /Na₂ CO₃ (10 mL)中之靈芝多醣體-F3(100 mg)與在DMF(1 mL)中之生物素醯胺基己醯基-6-胺基己酸N-羥基琥珀醯亞胺酯(生物素-XX-NHS)1.0 mg反應。將該混合物於室溫攪拌12小時。反應完成後，使用MWCO為6000-8000道爾頓之管狀膜於4℃透析48小時(5 x 500 mL)。透析後，將生物素基-F3冷凍乾燥，得到褐色粉末90 mg(90%)。生物素基-F3之純化藉由HPLC監測並使用鏈黴抗生物素蛋白-FITC進行結合檢定分析。Ganoderma lucidum polysaccharide-F3 was labeled with biotin in a "onepot" reaction. Specifically, Ganoderma lucidum polysaccharide-F3 (100 mg) in 0.2 N NaHCO ₃ /Na ₂ CO ₃ (10 mL) and biotin guanidinohexyl-6- in DMF (1 mL) Aminohexanoic acid N-hydroxysuccinimide (Biotin-XX-NHS) 1.0 mg reaction. The mixture was stirred at room temperature for 12 hours. After completion of the reaction, a tubular membrane having a MWCO of 6000-8000 Daltons was used for dialysis for 48 hours (5 x 500 mL) at 4 °C. After dialysis, biotinyl-F3 was freeze-dried to give a brown powder of 90 mg (90%). Purification of biotinyl-F3 was monitored by HPLC and assayed for binding assay using streptavidin-FITC.

實施例3：純化受體：Fc融合蛋白質之西方轉漬分析Example 3: Purification of Receptor: Western Transfer Analysis of Fc Fusion Protein

使實施例1之純化受體.Fc融合蛋白質進行電泳，移至硝基纖維素膜上(Hybond-C extra,Amersham Pharmacia Biotech)，並與(1：3000)之在TBST緩衝液(5%脫脂乾燥牛奶，在含有0.02% Tween 20之Tris緩衝鹽水中)中之綴合有過氧化酶之山羊抗人類IgG Ab(Jackson,PA,USA)反應。用TBST沖洗後，將吸漬物與強化化學發光試劑(Amersham Pharmacia Biotech)一起培育以顯現。The purified receptor.Fc fusion protein of Example 1 was subjected to electrophoresis, transferred to a nitrocellulose membrane (Hybond-C extra, Amersham Pharmacia Biotech), and (1:3000) in TBST buffer (5% degreased). The dried milk, reacted with peroxidase-conjugated goat anti-human IgG Ab (Jackson, PA, USA) in Tris buffered saline containing 0.02% Tween 20). After rinsing with TBST, the sorbate was incubated with an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech) for visualization.

實施例4：免疫吸附點結合法定分析Example 4: Immunoadsorption point combined with statutory analysis

生物素化F3於5倍系列稀釋後，使用Bio-Dot顯微過濾裝置TM(Bio-Rad,CA,USA)將其轉漬在經甲醇活化之PVDF膜(2 μL/點)上。在空氣中乾燥後，將轉漬片在TBST中培育，繼而與100 μL綴合有鏈黴抗生物素蛋白之辣根過氧化酶(HRP)(1：2000稀釋)(Chemicon,CA,USA)一起培育。結合反應用強化化學發光(ECL)試劑(Amersham Pharmacia Biotech)來顯現。Biotinylated F3 was transferred to a methanol activated PVDF membrane (2 μL/dot) using a Bio-Dot Microfiltration UnitTM (Bio-Rad, CA, USA) after 5 fold serial dilutions. After drying in air, the blots were incubated in TBST, followed by 100 μL of streptavidin-conjugated horseradish peroxidase (HRP) (1:2000 dilution) (Chemicon, CA, USA) Cultivate together. The binding reaction was visualized by an enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech).

亦將未生物素化之多醣體固定在經甲醇活化之PVDF膜上，繼而與100μL受體.Fc融合蛋白質(1 μg/ml，在2 mMCaCl₂ /TBST中)在Bio-Dot顯微過濾裝置TM(Bio-Rad,CA,USA)上於室溫培育1小時，然後與(1：3000)之在TBST緩衝液(5%脫脂乾燥牛奶，在含有0.02% Tween 20之Tris緩衝鹽水中)中之綴合有HRP之山羊抗-人類IgG抗體(Jackson,PA,USA)反應。用TBST沖洗後，將吸漬物與強化化學發光試劑(Amersham Pharmacia Biotech)一起培育以顯現。The unbiotinylated polysaccharide was also immobilized on a methanol-activated PVDF membrane, followed by 100 μL of receptor.Fc fusion protein (1 μg/ml in 2 mM CaCl ₂ /TBST) in a Bio-Dot microfiltration device. TM (Bio-Rad, CA, USA) was incubated for 1 hour at room temperature and then with (1:3000) in TBST buffer (5% degreased dry milk in Tris buffered saline containing 0.02% Tween 20) HRP-conjugated goat anti-human IgG antibody (Jackson, PA, USA) was conjugated. After rinsing with TBST, the sorbate was incubated with an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech) for visualization.

實施例5：重組受體.Fc融合蛋白質之表現Example 5: Recombinant Receptor. Expression of Fc Fusion Protein

來自免疫細胞之數種先天性免疫受體之細胞外區域依照實施例1之方法藉由反錄聚合酶連鎖反應(RT-PCR)選殖。將經擴增之DNA片段與pcDNA3/hIgG1-突變質體所含之人類IgG1之Fc部分融合。將經選殖之融合基因轉染入293 FREESTYLE哺乳動物細胞中，而分泌之蛋白質依照實施例1之方法藉由蛋白質珠粒純化。如圖1所示，選殖16種C-型凝集素基因(圖1A)。特定而言，圖1A顯示藉由RT-PCR擴增，然後在0.8%瓊脂糖上分成各部分並藉由溴化乙錠染色而顯現之先天性免疫受體之DNA片段。圖1B顯示在12%SDS-PAGE凝膠上電泳後之經表現之重組受體.Fc融合蛋白質。在圖1A及圖1B二者中，使用下列行之命名：第1行：CLEC2B/AICL，第2行：CLEC4C/BDCA-2，第3行：CLEC13A/BIMLEC，第4行：CLEC1A/CLEC-1，第5行：CLEC4D/CLEC-6，第6行：CLEC12A/CLL-1，第7行：CLEC4A/DCIR，第8行：CLEC4L/DC-SIGN，第9行：CLEC4M/DC-SIGNR，第10行：CLEC7A/樹狀細胞凝集素-1，第11行：CLEC6A/樹狀細胞凝集素-2，第12行：CLEC4H2/HBVxAgBP，第13行：CLEC4K/郎罕細胞特異蛋白(Langerin)，第14行：KLRG/MAFAL，第15行：DLVR1/CLEC5A(MDL-1)，第16行：CLEC4E/MINCLE。此外，亦以類似策略選殖及表現人類TREM(在骨髓細胞上表現之激發受體)-1、-2及類TREM轉錄體(TLT)-1、-2(Bouchon et al.,2000,J Immunol 164,4991-5；Daws et al.,2003,J Immunol 171,594-9；Washington et al.,2002,Blood 100,3822-4)。The extracellular regions of several innate immune receptors from immune cells were colonized by reverse transcription polymerase chain reaction (RT-PCR) according to the method of Example 1. The amplified DNA fragment was fused to the Fc portion of human IgG1 contained in the pcDNA3/hIgG1-mutaplast. The selected fusion gene was transfected into 293 FREESTYLE mammalian cells, and the secreted protein was purified by protein beads according to the method of Example 1. As shown in Figure 1, 16 C-type lectin genes were cloned (Fig. 1A). In particular, Figure 1A shows a DNA fragment of an innate immune receptor visualized by RT-PCR amplification followed by fractionation on 0.8% agarose and staining with ethidium bromide. Figure IB shows the expressed recombinant receptor.Fc fusion protein after electrophoresis on a 12% SDS-PAGE gel. In both Figures 1A and 1B, the following lines are used: Line 1: CLEC2B/AICL, Line 2: CLEC4C/BDCA-2, Line 3: CLEC13A/BIMLEC, Line 4: CLEC1A/CLEC- 1, line 5: CLEC4D/CLEC-6, line 6: CLEC12A/CLL-1, line 7: CLEC4A/DCIR, line 8: CLEC4L/DC-SIGN, line 9: CLEC4M/DC-SIGNR, Line 10: CLEC7A/dendritic cell lectin-1, line 11: CLEC6A/dendritic cell lectin-2, line 12: CLEC4H2/HBVxAgBP, line 13: CLEC4K/Langhan cell specific protein (Langerin) Line 14: KLRG/MAFAL, line 15: DLVR1/CLEC5A (MDL-1), line 16: CLEC4E/MINCLE. In addition, a similar strategy was used to select and display human TREM (excitation receptors expressed on bone marrow cells)-1, -2 and TREM-like transcripts (TLT)-1, -2 (Bouchon et al., 2000, J). Immunol 164, 4991-5; Daws et al., 2003, J Immunol 171, 594-9; Washington et al., 2002, Blood 100, 3822-4).

實施例6：固定之多醣體與受體.Fc融合蛋白質間之劑量依存***互作用Example 6: Dose-dependent interaction between immobilized polysaccharides and receptors. Fc fusion proteins

多醣體與受體.Fc融合蛋白質間之交互作用依照實施例4之方法使用點-結合檢定分析測試。靈芝多醣體之水溶性部分3(F3)(參見實施例3)含有活性成分以刺激產生細胞激素之細胞(Waang et al.,2002,Bioorg Med Chem 10,1057-62；Chen et al.,2004,Bioorg Med Chem 12,5595-601；Chien et al.,2004,Bioorg Med Chem 12,5603-9；Hsu et al.,2004,J Immunol 173,5989-99)。已知靈芝醣類含有具β-1,3-鍵結之多醣體骨架及具α-1,4-鍵結之聚甘露糖骨架中之任一者(Usui et al.,1983,Carbohydr.Res.,273；Miyazaki and Nishijime,1982,Carbohydr.Res.109,290；Bao et al.,2002,Phytochemistry 59,175-81)。已證明樹狀細胞凝集素-1(Dectin-1)受體(C型凝集素家族之成員)會與β-1,3-D-葡聚糖交互作用(Brown and Gordon,2001,Nature 413,36-7)。已證明樹狀細胞凝集素-1受體媒介β-葡聚糖之生物作用(Brown et al.,2003,J Exp Med 197,1119-24)。因此使用實施例4之點-結合檢定分析測試靈芝之F3部分是否會與樹狀細胞凝集素-1受體交互作用。Interaction between Polysaccharide and Receptor. Fc Fusion Protein The test was performed using the point-binding assay analysis according to the method of Example 4. The water-soluble fraction 3 (F3) of the Ganoderma lucidum polysaccharide (see Example 3) contains the active ingredient to stimulate cells producing cytokines (Waang et al., 2002, Bioorg Med Chem 10, 1057-62; Chen et al., 2004). , Bioorg Med Chem 12, 5595-601; Chien et al., 2004, Bioorg Med Chem 12, 5603-9; Hsu et al., 2004, J Immunol 173, 5989-99). It is known that Ganoderma lucidum saccharides contain either a β-1,3-bonded polysaccharide skeleton and an α-1,4-bonded polymannose skeleton (Usui et al., 1983, Carbohydr. Res). ., 273; Miyazaki and Nishijime, 1982, Carbohydr. Res. 109, 290; Bao et al., 2002, Phytochemistry 59, 175-81). The dendritic-1 (Dectin-1) receptor (a member of the C-type lectin family) has been shown to interact with β-1,3-D-glucan (Brown and Gordon, 2001, Nature 413, 36-7). The biological action of the dendritic cell lectin-1 receptor vector β-glucan has been demonstrated (Brown et al., 2003, J Exp Med 197, 1119-24). Therefore, the point-binding assay of Example 4 was used to test whether the F3 portion of Ganoderma lucidum interacts with the dendritic lectin-1 receptor.

將生物素化F3部分(圖2A中之“生物素-GLPS F3”)(依照實施例2製備)於5倍系列稀釋後固定在PVDF膜上並與綴合有鏈黴抗生物素蛋白之辣根過氧化酶(HRP)一起培育，再使用強化的化學發光試劑檢測生成之結合反應(參見實施例4)。如圖2A所示，點-結合檢定分析之靈敏度高於約0.08 μg。圖2A亦顯示當未生物素化之F3(圖2A中之“GLPS-F3”)固定在PVDF膜上，然後與綴合有鏈黴抗生物素蛋白之辣根過氧化酶(HRP)接觸時，未見到背景值。The biotinylated F3 fraction ("Biotin-GLPS F3" in Figure 2A) (prepared according to Example 2) was fixed on a PVDF membrane after 5 fold serial dilution and conjugated with streptavidin Roots were incubated with oxidase (HRP) and the resulting binding reaction was detected using a fortified chemiluminescent reagent (see Example 4). As shown in Figure 2A, the sensitivity of the point-binding assay is greater than about 0.08 μg. Figure 2A also shows that when unbiotinylated F3 ("GLPS-F3" in Figure 2A) is immobilized on a PVDF membrane and then contacted with horseradish peroxidase (HRP) conjugated with streptavidin No background value was seen.

將未生物素化之F3部分於系列稀釋後亦固定在PVDF膜上，並與100μL之1μg/mL樹狀細胞凝集素-1.Fc融合蛋白質或人類IgG1(做為陰性對照組)一起培育，繼而與綴合有HRP之山羊抗-人類IgG一起培育(參見實施例4)。如圖2B所示，樹狀細胞凝集素-1.Fc在點-結合檢定分析中可檢測到低於約1ng之F3之存在。在與人類IgG1而非樹狀細胞凝集素-1.Fc接觸之轉漬區無可見到之背景值。The unbiotinylated F3 fraction was also fixed on the PVDF membrane after serial dilution and incubated with 100 μL of 1 μg/mL dendritic lectin-1.Fc fusion protein or human IgG1 (as a negative control). This was followed by incubation with goat anti-human IgG conjugated with HRP (see Example 4). As shown in Figure 2B, dendritic cell lectin-1.Fc can detect the presence of less than about 1 ng of F3 in a point-binding assay. There were no background values visible in the transfected area in contact with human IgG1 but not dendritic lectin-1.Fc.

圖2B之轉漬片(blot)之點密度藉由密度計(ImageQuant)測定，而該結果顯示樹狀細胞凝集素-1.Fc結合信號以劑量依存方式增加(參見圖2C)。The dot density of the blot of Figure 2B was determined by a densitometer (ImageQuant), and the results showed that the dendritic cell lectin-1.Fc binding signal increased in a dose-dependent manner (see Figure 2C).

為了測定其他多醣體是否抑制F3與樹狀細胞凝集素-1間之交互作用，將F3(10μg/點)固定在PVDF膜上，然後於β-葡聚糖、D-葡萄糖及D-半乳糖(0.1μg-1000μg)之系列稀釋溶液存在下，與100 μL樹狀細胞凝集素-1.Fc(1μg/mL)接觸，繼而與綴合有HRP之山羊抗-人類IgG一起培育。圖2D顯示就競爭者β-葡聚糖而言之轉漬片之點密度，圖2E顯示所有競爭者之轉漬像。由此等可見到樹狀細胞凝集素-1.Fc與F3部分間之交互作用被β-1,3-葡聚糖抑制，但不會被D-葡萄糖或D-半乳糖抑制。此表示樹狀細胞凝集素-1.Fc與F3間之交互作用係經由β-1,3-葡聚糖識別。In order to determine whether other polysaccharides inhibit the interaction between F3 and dendritic cell lectin-1, F3 (10 μg/dot) was immobilized on PVDF membrane, followed by β-glucan, D-glucose and D-galactose. In the presence of a serial dilution solution (0.1 μg - 1000 μg), it was contacted with 100 μL of dendritic cell lectin-1.Fc (1 μg/mL), and then incubated with goat anti-human IgG conjugated with HRP. Figure 2D shows the dot density of the rotating tablets for competitor β-glucan, and Figure 2E shows the image of all competitors. Thus, it can be seen that the interaction between the dendritic cell lectin-1.Fc and the F3 moiety is inhibited by β-1,3-glucan, but is not inhibited by D-glucose or D-galactose. This indicates that the interaction between dendritic cell lectin-1.Fc and F3 is recognized via β-1,3-glucan.

實施例7：能與F3部分交互作用之受體之鑑定Example 7: Identification of receptors capable of interacting with the F3 moiety

檢定分析F3與C-型凝集素家族之其他成員或與類Ig受體之交互作用。將未生物素化之F3及未生物素化之F3C(衍生自通過100 kDa MWCO離心管之F3)(10μg/點)固定在PVDF膜上(參見實施例4)，然後與100μL之25種不同重組受體.Fc融合蛋白質(包括19種凝集素受體及8個TREM及TLT家族成員)以及做為對照之人類IgG1的1μg/mL溶液一起培育。使用綴合有HRP之山羊抗-IgG抗體及ECL試劑檢測結合。結果以表格形式示於圖3中(相對點強度以“+”記號表示，而未檢測到結合以“-“記號表示)而轉漬片之影像示於圖4A中。圖3中之探針編號系統保留於圖4A中。The assay analyzes the interaction of F3 with other members of the C-type lectin family or with Ig-like receptors. Unbiotinylated F3 and unbiotinylated F3C (derived from F3 through a 100 kDa MWCO centrifuge tube) (10 μg/dot) were mounted on a PVDF membrane (see Example 4) and then different from 25 μL of 100 μL Recombinant receptor. Fc fusion proteins (including 19 lectin receptors and 8 TREM and TLT family members) were incubated with a 1 [mu]g/mL solution of human IgGl as a control. Binding was detected using a goat anti-IgG antibody conjugated to HRP and an ECL reagent. The results are shown in tabular form in Fig. 3 (the relative dot intensity is indicated by a "+" mark, and the undetected binding is indicated by a "-" mark) and the image of the transferred patch is shown in Fig. 4A. The probe numbering system of Figure 3 is retained in Figure 4A.

此等結果顯示除了樹狀細胞凝集素-1.Fc(圖3及圖4A中之14號探針)之外，F3亦與KCR.Fc(圖3及圖4A中之7號探針)、DC-SIGNR.Fc(圖3及圖4A中之11號探針)及TLT-2.Fc(圖3及圖4A中之21號探針)交互作用。有趣的是，F3C(其係衍生自通過100 kDa MWCO離心管之F3)卻具有較低之對於TLT2之結合親和力。此暗示TLT2可區辨F3與F3c間之微小差異。These results show that in addition to dendritic lectin-1.Fc (probe 14 of Figure 3 and Figure 4A), F3 is also associated with KCR.Fc (probe 7 of Figure 3 and Figure 4A), DC-SIGNR.Fc (probe 11 of Figures 3 and 4A) and TLT-2.Fc (probe 21 of Figure 3 and Figure 4A) interact. Interestingly, F3C, which is derived from F3 through a 100 kDa MWCO centrifuge tube, has a lower binding affinity for TLT2. This suggests that TLT2 can distinguish between small differences between F3 and F3c.

凝集素受體家族成員在交互作用上依存於Ca⁺⁺ ；所以研究EDTA(乙二胺四乙酸)抑制與F3結合之能力。已發現EDTA(10mM，在TBST中)完全去除F3與KCR.Fc及與DC-SIGNR.Fc之交互作用，但不會去除F3與樹狀細胞凝集素-1.Fc及TLT2.Fc之交互作用。圖4B係圖示存在及不存在Ca⁺⁺ 下所製得之轉漬片之影像(左格只有TBST；右格為10mM EDTA+TBST)。使用綴合有HRP之山羊抗-IgG抗體及ECL試劑檢測結合。該結果與先前觀察到之配體與KCR間之交互作用一致(Hoyle and Hill,1988,J Biol Chem 263,7487-92)，且DC-SIGNR為Ca⁺⁺ -依存性(Soilleux et al.,2000,J Immunol 165,2937-42)，而Ca⁺⁺ 對於樹狀細胞凝集素-1與β-1,3-葡聚糖間之交互作用而言非為必要(Herre et al.,2004,Mol Immunol 40,869-76)。因此，F3顯然含有豐富的葡聚糖，其可與免疫細胞上之多個受體同時交互作用。Members of the lectin receptor family depend on Ca ^{++ for} interaction; therefore, the ability of EDTA (ethylenediaminetetraacetic acid) to inhibit binding to F3 was investigated. It has been found that EDTA (10 mM in TBST) completely removes the interaction of F3 with KCR.Fc and with DC-SIGNR.Fc, but does not remove the interaction of F3 with dendritic lectin-1.Fc and TLT2.Fc. . Figure 4B is an image showing the presence and absence of a rotating patch prepared under Ca ⁺⁺ (left only TBST; right grid is 10 mM EDTA + TBST). Binding was detected using a goat anti-IgG antibody conjugated to HRP and an ECL reagent. This result is consistent with the previously observed interaction between the ligand and KCR (Hoyle and Hill, 1988, J Biol Chem 263, 7487-92), and DC-SIGNR is Ca ⁺⁺ -dependent (Soilleux et al., 2000, J Immunol 165, 2937-42), and Ca ⁺⁺ is not necessary for the interaction between dendritic lectin-1 and β-1,3-glucan (Herre et al., 2004, Mol Immunol 40, 869-76). Therefore, F3 is clearly rich in glucan, which can interact simultaneously with multiple receptors on immune cells.

圖4C圖示使用β-葡聚糖做為多醣體(10μg/點)及使用100μL之1μg/mL樹狀細胞凝集素-1.Fc、DC-SIGN.Fc、mKCR.Fc及TLT2.Fc之轉漬點。使用綴合有HRP之山羊抗-IgG抗體及ECL試劑檢測結合。四種受試之受體.Fc融合蛋白質中，只有樹狀細胞凝集素-1.Fc可與β-1,3-葡聚糖結合。此表示其他三種受體.Fc融合蛋白質結合於β-1,3-葡聚糖以外之糖成分。4C shows the use of β-glucan as a polysaccharide (10 μg/dot) and 100 μL of 1 μg/mL dendritic cell lectin-1.Fc, DC-SIGN.Fc, mKCR.Fc and TLT2.Fc Turn the stain point. Binding was detected using a goat anti-IgG antibody conjugated to HRP and an ECL reagent. Among the four receptor-Fc fusion proteins tested, only dendritic lectin-1.Fc binds to β-1,3-glucan. This indicates that the other three receptors. The Fc fusion protein binds to a sugar component other than β-1,3-glucan.

實施例8：來自各種來源之多醣體之獨特細微特徵Example 8: Unique subtle features of polysaccharides from various sources (fingerprints)(fingerprints)

用樹狀細胞凝集素-1.Fc、mKCR.Fc、DC-SIGNR.Fc及TLT2.Fc融合蛋白質進行實施例4之點-結合檢定分析，以指紋辨識單離自冬蟲夏草及市場上之其他來源之多醣體之獨特細微特徵。如上所述將各多醣體組合物固定在PVDF膜上，然後與100μL之1μg/mL融合蛋白質溶液接觸。使用綴合有HRP之山羊抗-IgG抗體及ECL試劑檢測結合。圖5A顯示各融合蛋白質之個別轉漬點而圖5B以表格形式顯示樣品編號及相對點強度。靈芝粗製萃取物(圖5中之5號點)只與樹狀細胞凝集素-1.Fc及DC-SIGNR.Fc交互作用，而來自粗製萃取物之純化F3(1號點)與所有四種受體交互作用。此表示F3純化過程可使能與免疫受體交互作用之成分富化。來自冬蟲夏草之多醣體(7號點)會與樹狀細胞凝集素-1.Fc強力地交互作用，此表示該多醣體含有β-1,3葡聚糖，但其與其他三種受體之交互作用與F3相較則弱許多。單離自霍山石斛之多醣體在碘試驗反應(見實施例2)中為陽性，此暗示此等部分主要包含α-D-葡聚糖。與單離自真菌者相反，霍山石斛之多醣體之混合物(6號點)不會與四種受體.Fc融合蛋白質之任一種反應。藉由ddH₂ O自蘑菇多醣體單離之多醣體(部分L，8號點)及用0.25N NaOH自蘑菇多醣體單離而得之多醣體(部分M，9號點)與樹狀細胞凝集素-1.Fc及DC-SIGNR.Fc之結合程度不同。因此，該法可查明單離自不同來源及製備之多醣體之獨特細微特徵(fingerprints)。Point-binding assay analysis of Example 4 was performed using dendritic cell lectin-1.Fc, mKCR.Fc, DC-SIGNR.Fc and TLT2.Fc fusion proteins, fingerprinting alone from Cordyceps sinensis and other sources on the market The unique and subtle features of the polysaccharide. Each polysaccharide composition was immobilized on a PVDF membrane as described above, and then contacted with 100 μL of a 1 μg/mL fusion protein solution. Binding was detected using a goat anti-IgG antibody conjugated to HRP and an ECL reagent. Figure 5A shows individual blotting points for each fusion protein and Figure 5B shows sample numbers and relative dot intensities in tabular form. Ganoderma lucidum crude extract (point 5 in Figure 5) interacts only with dendritic lectin-1.Fc and DC-SIGNR.Fc, while purified F3 (point 1) from crude extract and all four Receptor interaction. This means that the F3 purification process enables enrichment of components that interact with the immune receptor. The polysaccharide from Cordyceps sinensis (point 7) interacts strongly with dendritic cell lectin-1.Fc, which means that the polysaccharide contains beta-1,3 glucan, but its interaction with the other three receptors The effect is much weaker than that of F3. The polysaccharides isolated from Dendrobium huoshanense were positive in the iodine test reaction (see Example 2), suggesting that these moieties mainly comprise α-D-glucan. In contrast to the isolates from the fungus, the mixture of polysaccharides of Dendrobium huoshanense (point 6) does not react with any of the four receptor.Fc fusion proteins. Polysaccharide (part L, No. 8) isolated from mushroom polysaccharide by ddH ₂ O and polysaccharide (part M, 9) and dendritic cells isolated from mushroom polysaccharide with 0.25N NaOH The degree of binding of lectin-1.Fc and DC-SIGNR.Fc is different. Thus, the method can identify unique fingerprints that are isolated from different sources and prepared polysaccharides.

實施例6-8說明F3與樹狀細胞凝集素-1.Fc、mKCR.Fc、DC-SIGNR.Fc及TLT2.Fc之交互作用。庫佛氏(Kupffer)細胞受體(KCR)對於D-半乳糖及N-乙醯基半乳糖胺具有高親和力(Fadden et al.,2003,Glycobiology 13,529-37)，並能清除血清之以D-半乳糖或D-岩藻糖為終端之醣蛋白(Lehrman et al.,1986,J Biol Chem 261,7426-32)。F3之免疫調節功能依存於岩藻糖之存在，且藉由α1,2-岩藻糖苷酶進行之醣裂解會使F3失去活性。因此該四種受體是否可與醣裂解後之F3交互作用令人感到興趣。DC-SIGNR/L-SIGN在結構上與DC-SIGN近似(77%相同)，但其只在肝竇、淋巴結及胎盤之內皮細胞中表現(Van Liempt et al.,2004,J BiOl Chem 279,33161-7)。DC-SIGN及DC-SIGNR二者皆可與N-鍵結有高量甘露糖之寡糖(Man₉ GlcNAc₂ Asn醣肽)結合。不過，只有DC-SIGN可與具終端岩藻糖殘基之聚醣結合，而DC-SIGNR則否(Guo et al.,2004,Nat Struct Mol Biol 11,591-8)。縱使與DC-SIGN相較，DC-SIGNR係與較窄範圍之配體結合，但只有DC-SIGNR可與F3交互作用。此暗示F3可能含有與Fucα1-4GlcNAc、Lewis^X 、Lewis^a 及血型糖抗原決定部位(blood group sugar epitopes)(DC-SIGN之已知配體)不同之獨特結構。Examples 6-8 illustrate the interaction of F3 with dendritic cell lectin-1.Fc, mKCR.Fc, DC-SIGNR.Fc and TLT2.Fc. Kupffer Cell Receptor (KCR) has high affinity for D-galactose and N-ethylmercaptogalactosamine (Fadden et al., 2003, Glycobiology 13, 529-37) and is capable of clearing serum to D - Galactose or D-fucose is the terminal glycoprotein (Lehrman et al., 1986, J Biol Chem 261, 7426-32). The immunomodulatory function of F3 is dependent on the presence of fucose, and saccharide cleavage by α1,2-fucosidase deactivates F3. It is therefore of interest whether the four receptors can interact with F3 after sugar cleavage. DC-SIGNR/L-SIGN is structurally similar to DC-SIGN (77% identical), but it only appears in endothelial cells of the hepatic sinus, lymph nodes, and placenta (Van Liempt et al., 2004, J BiOl Chem 279, 33161-7). Both DC-SIGN and DC-SIGNR can bind to N-linked mannose-rich oligosaccharides (Man ₉ GlcNAc ₂ Asn glycopeptide). However, only DC-SIGN can bind to glycans with terminal fucose residues, whereas DC-SIGNR does not (Guo et al., 2004, Nat Struct Mol Biol 11, 591-8). Even though DC-SIGNR is combined with a narrower range of ligands than DC-SIGN, only DC-SIGNR can interact with F3. This suggests that F3 may contain ^a unique structure different from Fucα1-4GlcNAc, Lewis ^X , Lewis ^a, and blood group sugar epitopes (known ligands for DC-SIGN).

TLT-2為類TREM轉錄體家族之成員，其含有獨特的單一V-組免疫球蛋白(Ig)區域及細胞質長尾部，該細胞質長尾部含脯胺酸富化區及免疫受體酪胺酸系抑制性基序(immune receptor tyrosine-based inhibitory motif，ITIM)，已知該ITIM用於與蛋白質酪胺酸磷酸酶之交互作用(Washington et al.,2002,Blood 100,3822-4；Washington et al.,2004,Blood 104,1042-7)。既然F3具有強力的免疫刺激功能，因而在未來研究是否藉由親和性層析從F3移除TLT2.Fc.結合性成分可進一步增強F3之刺激功能將可能為有趣的。或者可藉由使用樹狀細胞凝集素-1.Fc、KCR.Fc及DC-SIGNR.Fc進行親和性層析進一步純化F3，以移除F3中之其他成分。TLT-2 is a member of the TREM-like transcript family, which contains a unique single V-group immunoglobulin (Ig) region and a long cytoplasmic tail. The long tail contains a proline-rich region and an immunoreceptor tyrosine. Immune receptor tyrosine-based inhibitory motif (ITIM), which is known to interact with protein tyrosine phosphatase (Washington et al., 2002, Blood 100, 3822-4; Washington et al) Al., 2004, Blood 104, 1042-7). Since F3 has potent immunostimulatory functions, it may be interesting to investigate whether it is possible to further enhance the stimulation function of F3 by removing the TLT2.Fc. binding component from F3 by affinity chromatography in the future. Alternatively, F3 can be further purified by affinity chromatography using dendritic lectin-1.Fc, KCR.Fc and DC-SIGNR.Fc to remove other components of F3.

F3與F3c之間、F3與靈芝1-3之間、以及蘑菇多醣體部分L及M之間之微細差異暗示本文所例示之此四種受體.Fc融合蛋白質可被用於使純化步驟達最佳化並監測來自不同來源或來自不同發酵條件之多醣體之差異。The slight difference between F3 and F3c, between F3 and Ganoderma lucidum 1-3, and between the mushroom polysaccharide fractions L and M suggests that the four receptors Fc fusion proteins exemplified herein can be used to make the purification step up. The differences in polysaccharides from different sources or from different fermentation conditions are optimized and monitored.

實施例9：可與GLPS-F3交互作用之人類凝集素受體在微量滴定平皿上藉由酶鍵連免疫檢定分析之鑑定Example 9: Identification of Human Lectin Receptors Interacting with GLPS-F3 by Microtiter Plates by Enzyme-Linked Immunoassay

多醣體與受體.Fc融合蛋白質間之交互作用藉由進行酶-鍵連免疫檢定分析(EIA)而進一步研究，其係依據將GLPS-F3經由親水力量及疏水力量固定於微量滴定平皿(聚苯乙烯)上。以此方式，供剖析之不同受體.Fc融合蛋白質之數目與實施例7相較增加。為了使GLPS-F3之固定量最佳化，將各種量(3-1000ng/孔，用100mM Tris緩衝液(pH9.5)稀釋)之生物素化GLPS-F3(生物素-GLPS-F3)塗佈在MaxiSorp StarWell微量滴定平皿(50μL/孔；Nunc)上。將該等平皿於4℃培育整夜，然後將此等孔用TBST沖洗二次，繼而用200 μL封阻緩衝液(2% BSA/TBST)於室溫封阻1小時。然後使用綴合有過氧化酶之抗生物素蛋白(稀釋度1：5000，Vector Laboratories)及TMB(四甲基聯苯胺)受質來檢測經固定之生物素化GLPS-F3。如圖6A所示，塗佈平皿之生物素-GLPS-F3之量為100 ng/孔時達到恆穩狀態，所以在EIA中選用該量進行未生物素化之GLPS-F3之固定。The interaction between the polysaccharide and the receptor.Fc fusion protein was further studied by performing enzyme-linked immunoassay (EIA) based on immobilization of GLPS-F3 to the microtiter plate via hydrophilic force and hydrophobic force. Styrene). In this way, the number of different receptor.Fc fusion proteins for profiling was increased as compared to Example 7. In order to optimize the fixed amount of GLPS-F3, various amounts (3-1000 ng/well, diluted with 100 mM Tris buffer (pH 9.5)) biotinylated GLPS-F3 (Biotin-GLPS-F3) were applied. The cloth was placed on a MaxiSorp StarWell microtiter plate (50 μL/well; Nunc). The plates were incubated overnight at 4 ° C, then the wells were rinsed twice with TBST and then blocked with 200 μL of blocking buffer (2% BSA/TBST) for 1 hour at room temperature. The immobilized biotinylated GLPS-F3 was then tested using a peroxidase-conjugated avidin (dilution 1:5000, Vector Laboratories) and TMB (tetramethylbenzidine). As shown in Fig. 6A, the amount of biotin-GLPS-F3 coated on the plate reached a constant state at 100 ng/well, so this amount was selected in EIA for immobilization of unbiotinylated GLPS-F3.

然後測試GLPS-F3與受體.Fc間之交互作用。如上所述以100 ng/孔將未生物素化之GLPS-F3固定，再將100μL受體.Fc融合蛋白質(1 μg/ml，在2 mM MgCl₂ /2 mM CaCl₂ /1% BSA/TBST中)加至各孔中並於室溫培育1小時。用TBST沖洗後，將此等孔與綴合有過氧化酶之山羊抗-人類IgG Ab在封阻緩衝液中於室溫一起培育30分鐘(稀釋度為1：5000，Jackson ImmunoResearch Laboratories)。此等孔用TBST沖洗後，與100 μL TMB受質一起培育15分鐘並在融合平皿讀數器(Perkin Elmer)中於450 nm測量。將結果用Fc.樹狀細胞凝集素-1結合度為基準予以歸一化(樹狀細胞凝集素-1為已知能與β-1,3-葡聚糖結合之凝集素受體，該β-1,3-葡聚糖為在GLPS-F3中所發現之骨架)。圖6B圖示相較於樹狀細胞凝集素-1，各受體對於GLPS-F3之親和力。結果顯示觀察到Fc.郎罕細胞特異蛋白、Fc.DC-SIGN、MMR.Fc、TLR2.Fc、TLR4.Fc、Fc.CLEC-2(CLEC1B)及Fc.CLEC-6(CLEC4D)對於GLPS-F3有高親和力(在本檢定分析中，高親和力被定義與Fc.樹狀細胞凝集素-1相較，結合強度>50%)。值得注意的是TLR2及TLR4[已證明其在GLPS-誘生之細胞活化上扮有角色(Hsu et al.,J Immunol 173：5989-5999(2004)；Shao et al.,Biochem Biophys Res Commun 323：133-141(2004))]在EIA中亦會與GLPS-F3結合。在Fc.NKG2D、Fc.MINCLE、Fc.mKCR、DCAL1.Fc、DEC205.Fc、Endo180.Fc及NKp30(NCR3).Fc中亦發現較弱，但為陽性之GLPS-F3結合能力(與Fc.樹狀細胞凝集素-1相較，結合強度為25-50%)。其他凝集素受體，包括Fc.AICL、Fc.BDCA2、Fc.CLEC1、Fc.CLL1、Fc.DCIR、Fc.DC-SIGNR、Fc.樹狀細胞凝集素-2、Fc.MDL-1及Fc.ML2，如同對照組人類IgG1，對於GLPS-F3具有極小的結合能力。The interaction between GLPS-F3 and the receptor.Fc was then tested. Unbiotinylated GLPS-F3 was immobilized at 100 ng/well as described above, and then 100 μL of receptor.Fc fusion protein (1 μg/ml in 2 mM MgCl ₂ /2 mM CaCl ₂ /1% BSA/TBST Medium) was added to each well and incubated for 1 hour at room temperature. After washing with TBST, the wells were incubated with goat anti-human IgG Ab conjugated with peroxidase in blocking buffer for 30 minutes at room temperature (dilution 1:5000, Jackson ImmunoResearch Laboratories). These wells were rinsed with TBST, incubated with 100 μL of TMB substrate for 15 minutes and measured at 450 nm in a fused plate reader (Perkin Elmer). The results were normalized based on the binding degree of Fc. dendritic cell lectin-1 (dendritic cell lectin-1 is a lectin receptor known to bind to β-1,3-glucan, which is β - 1,3-glucan is the backbone found in GLPS-F3). Figure 6B shows the affinity of each receptor for GLPS-F3 compared to dendritic cell lectin-1. The results showed that Fc. Langhan cell-specific protein, Fc.DC-SIGN, MMR.Fc, TLR2.Fc, TLR4.Fc, Fc.CLEC-2 (CLEC1B) and Fc.CLEC-6 (CLEC4D) were observed for GLPS- F3 has high affinity (in this assay, high affinity is defined compared to Fc. dendritic cell lectin-1, binding strength >50%). Of note, TLR2 and TLR4 [have been shown to play a role in GLPS-induced cell activation (Hsu et al., J Immunol 173: 5989-5999 (2004); Shao et al., Biochem Biophys Res Commun 323 :133-141 (2004))] will also be combined with GLPS-F3 in EIA. A weaker but positive GLPS-F3 binding capacity was also found in Fc.NKG2D, Fc.MINCLE, Fc.mKCR, DCAL1.Fc, DEC205.Fc, Endo180.Fc and NKp30(NCR3).Fc (with Fc. Compared with dendritic cell lectin-1, the binding strength is 25-50%). Other lectin receptors, including Fc.AICL, Fc.BDCA2, Fc.CLEC1, Fc.CLL1, Fc.DCIR, Fc.DC-SIGNR, Fc. dendritic cell agglutinin-2, Fc.MDL-1 and Fc .ML2, like the control human IgG1, has minimal binding capacity for GLPS-F3.

實施例10：與先天性免疫受體交互作用之GLPS-F3之競爭性檢定分析Example 10: Competitive assay analysis of GLPS-F3 interacting with innate immune receptors

為了解GLPS-F3與特異性先天性免疫受體之交互作用，將多醣體甘露聚糖及β-葡聚糖以及單醣體D-甘露糖(Man)、D-葡萄糖(Glc)、N-乙醯基-葡萄糖胺(GlcNAc)、D-半乳糖(Gal)、N-乙醯基-半乳糖胺(GalNAc)、L-岩藻糖(Fuc)及唾液酸用於競爭性檢定分析。檢定對於GLPS-F3顯示較高結合能力之先天性免疫受體，包括Fc.樹狀細胞凝集素-1、Fc.郎罕細胞特異蛋白、Fc.DC-SIGN、TLR4.Fc、MMR.Fc、Fc.CLEC-2(CLEC1B)及Fc.CLEC-6(CLEC4D)。檢定分析如同實施例9進行，其中加入1mg/ml之各多醣體或單醣體。To understand the interaction between GLPS-F3 and specific innate immune receptors, polysaccharide mannan and β-glucan as well as monosaccharides D-mannose (Man), D-glucose (Glc), N- Ethyl glucosamine (GlcNAc), D-galactose (Gal), N-ethylmercapto-galactosamine (GalNAc), L-fucose (Fuc) and sialic acid were used for competitive assay analysis. Innate immune receptors showing high binding capacity for GLPS-F3, including Fc. dendritic cell lectin-1, Fc. Langer cell-specific protein, Fc.DC-SIGN, TLR4.Fc, MMR.Fc, Fc.CLEC-2 (CLEC1B) and Fc.CLEC-6 (CLEC4D). The assay was performed as in Example 9, in which 1 mg/ml of each polysaccharide or monosaccharide was added.

如圖7(圖示各受體/醣組合，相對於醣不存在時所見到之結合度之結合%)及表1(以表格形式提供來自圖7之數據)所示，GLPS-F3與Fc.樹狀細胞凝集素-1間之交互作用可被β-葡聚糖封阻，其抑制度為58%，此符合已發表之結果(Palma et al.,J Biol Chem 281：5771-5779(2006)；Willment et al.,J Biol Chem 276：43818-43823(2001))。唾液酸之加入(抑制度83%)會干擾Fc.樹狀細胞凝集素-1與GLPS-F3之結合。Fc.郎罕細胞特異蛋白與GLPS-F3間之交互作用會被甘露聚糖、Man及GlcNAc阻擾(抑制度分別為95%,26%及84%)，據報告該等為郎罕細胞特異蛋白之糖配體(Stambach & Taylor,Glycobiology 13：401-410(2002))；亦觀察到唾液酸(抑制度為95%)會干擾Fc.郎罕細胞特異蛋白與GLPS-F3之結合。至於Fc.DC-SIGN與GLPS-F3之結合，甘露聚糖、Man、Fuc及唾液酸顯示強力的封阻活性(抑制度98%,72%,92%及90%)，而Glc及GlcNAc在封阻交互作用上具有較弱的作用(抑制度分別為45%及27%)。甘露聚糖、Man、Glc、GlcNAc、Gal、Fuc及唾液酸封阻GLPS-F3與MMR.Fc間之交互作用(抑制度分別為98%,87%,45%,78%,36%,88%及93%)，其中該MMR.Fc已知為會與Man、Fuc、GlcNAc及唾液基Lewis x(sLex)結合之重要凝集素受體(Letuex et al.,J Exp Med 191：1117-1126(2000)；Stahl,Am J Respir Cell Mol Biol 2：317-318(1990))。Fc.CLEC-2與GLPS-F3間之交互作用會藉由加入唾液酸而封阻(抑制度55%)。對Fc.CLEC-6而言，未觀察到受試之糖顯示明顯之封阻作用。顯然地，甘露聚糖及Fuc對於TLR4.Fc與GLPS-F3之交互作用顯示封阻作用(抑制度分別為72%及44%)。此處得到之數據與對於樹狀細胞凝集素-1、郎罕細胞特異蛋白、DC-SIGN及MMR所報告之糖配體研究之結果一致。亦顯示許多凝集素受體可經由不同的糖成分以多價與GLPS-F3結合。Figure 7 (showing the % binding of each receptor/sugar combination relative to the degree of binding seen in the absence of sugar) and Table 1 (providing the data from Figure 7 in tabular form), GLPS-F3 and Fc The interaction between dendritic cell lectin-1 can be blocked by β-glucan with a degree of inhibition of 58%, which is consistent with published results (Palma et al., J Biol Chem 281:5771-5779 ( 2006); Willment et al., J Biol Chem 276:43818-43823 (2001)). The addition of sialic acid (83% inhibition) interferes with the binding of Fc. dendritic lectin-1 to GLPS-F3. The interaction between Fc. Langhan cell-specific protein and GLPS-F3 was blocked by mannan, Man and GlcNAc (inhibition degrees 95%, 26% and 84%, respectively), which were reported to be Langer cell-specific Glycoprotein ligands (Stambach & Taylor, Glycobiology 13: 401-410 (2002)); sialic acid (95% inhibition) was also observed to interfere with the binding of Fc. Langhan cell-specific proteins to GLPS-F3. As for the combination of Fc.DC-SIGN and GLPS-F3, mannan, Man, Fuc and sialic acid showed potent blocking activity (inhibition 98%, 72%, 92% and 90%), while Glc and GlcNAc were The blocking interaction has a weaker effect (inhibition degrees of 45% and 27%, respectively). Mannan, Man, Glc, GlcNAc, Gal, Fuc and sialic acid blocked the interaction between GLPS-F3 and MMR.Fc (inhibition degrees were 98%, 87%, 45%, 78%, 36%, 88, respectively). % and 93%), wherein the MMR.Fc is known to be an important lectin receptor that binds to Man, Fuc, GlcNAc and salivary Lewis x (sLex) (Letuex et al., J Exp Med 191:1117-1126) (2000); Stahl, Am J Respir Cell Mol Biol 2:317-318 (1990)). The interaction between Fc.CLEC-2 and GLPS-F3 is blocked by the addition of sialic acid (55% inhibition). For Fc.CLEC-6, no detectable sugar showed significant blocking effect. Apparently, the interaction of mannan and Fuc on TLR4.Fc and GLPS-F3 showed a blocking effect (inhibition levels of 72% and 44%, respectively). The data obtained here are consistent with the results of studies on glycoconjugates reported for dendritic cell lectin-1, Langerhans cell specific protein, DC-SIGN and MMR. It has also been shown that many lectin receptors can bind to GLPS-F3 in multiple ways via different sugar components.

表1：於存在糖競爭者下，先天性免疫受體.Fc融合蛋白質與CLPS-F3之結合度，相對於不存在糖競爭者下所見到之結合度之百分率。Table 1: Percentage of binding of the innate immune receptor. Fc fusion protein to CLPS-F3 in the presence of a sugar competitor, relative to the degree of binding seen in the absence of a sugar competitor.

實施例7至10所呈示之系統為以高通量剖析(profiling)GLPS以及其他醣蛋白混合物(包括目前使用之許多中藥)之有用工具。藉由使用不同表面(PVDF及聚苯乙烯)進行多醣體之固定，得到GLPS-F3的不同剖析結果。此可能係由於混合物中某些多醣體偏好結合於不同表面。從該二互補形式(format)得到之結果提供多醣體混合物之「指紋(獨特細微特徵)」。可將此等指紋辨識多醣體混合物之獨特細微特徵之方法用於，例如，監測在不同條件下，來自不同來源或來自不同批號之草藥萃取物之含量。再者，從特定多醣體混合物之剖析收集到之資料在了解其於活體內之生物作用之分子機制上具有重要性。The systems presented in Examples 7 through 10 are useful tools for profiling GLPS and other glycoprotein mixtures, including many of the traditional Chinese medicines currently in use, with high throughput. Different profiling results of GLPS-F3 were obtained by immobilizing polysaccharides using different surfaces (PVDF and polystyrene). This may be due to the preference of certain polysaccharides in the mixture to bind to different surfaces. The results obtained from the two complementary forms provide a "fingerprint (unique subtle feature)" of the polysaccharide mixture. The method of fingerprinting the unique subtle features of the polysaccharide mixture can be used, for example, to monitor the amount of herbal extracts from different sources or from different batches under different conditions. Furthermore, the data collected from the profiling of a particular polysaccharide mixture is important in understanding the molecular mechanisms by which it acts in vivo.

實施例11:DVLR1/CLEC5A(MDL-1)與登登革熱病毒之交互作用之檢測Example 11: Detection of interaction between DVLR1/CLEC5A (MDL-1) and dengue virus

下列實施例顯示本發明之融合蛋白質及方法如何用於鑑定可與病原體交互作用之先天性免疫受體，以及該等資料如何隨後用於測定與先天性免疫受體結合之病原體之下游作用，並亦用於設計治療病原體感染之治療劑。The following examples demonstrate how the fusion proteins and methods of the invention can be used to identify innate immune receptors that interact with pathogens, and how such data can subsequently be used to determine the downstream effects of pathogens that bind to innate immune receptors, and It is also used to design therapeutic agents for the treatment of pathogen infections.

登革熱為侵犯人類之最重要蚊傳病毒疾病之一。其全球分布與瘧疾之分布相當，且據估計活在有流行傳染之虞地區之人達2.5億。登革熱病毒(DV)感染後之臨床症候群包括登革熱(DF)及出血性登革熱(DHF)/登革熱休克症候群(DSS)。不過，導致DHF及DSS之分子機制仍未獲得充分釐清。Dengue fever is one of the most important mosquito-borne diseases that invade humans. Its global distribution is comparable to the distribution of malaria, and it is estimated that there are 250 million people living in areas with epidemic infections. Clinical syndromes following dengue virus (DV) infection include dengue fever (DF) and hemorrhagic dengue fever (DHF)/dengue shock syndrome (DSS). However, the molecular mechanisms leading to DHF and DSS have not yet been fully clarified.

已知DC-SIGN媒介人類樹狀細胞之登革熱(DV)感染(Tassaneetrithepet al.,J Exp Med,2003.197(7)：p.823-9)。為了解DV之致病原因，測定DV是否與來自樹狀細胞、巨噬細胞、天然殺手細胞及周圍血液單核細胞(PBMCs)之其他膜-結合性C-型凝集素受體及類C型凝集素受體交互作用頗為重要。為達成此，將DVLR1/CLEC5A(MDL-1)、樹狀細胞凝集素-1、KCR及DC-SIGN(做為陽性對照阻)之細胞外區域與人類IgG1之Fc部分融合。更特定而言，使用DC-SIGN之引子(序列編號：17及序列編號：18)、DVLR1/CLEC5A之引子(序列編號：21及序列編號：22)、樹狀細胞凝集素-1之引子(序列編號：25及序列編號：26)以及KCR之引子(前向：5'-CAGCCTTGGAGACCTGAGT-3'序列編號：37；逆向5'-TAGCCTACTCTGGCCGC-3’序列編號：38)產生經擴增之cDNA片段。各前向引子具有額外的BamH1部位，且各逆向引子具有額外的EcoRI部位，以加速經擴增之cDNA次選殖入含人類IgG1 Fc部分之pCDNA3.1(Invitrogen)哺乳動物表現載體。然後將生成之載體轉染入293 FreeStyle細胞(lnvitrogen)，以產生可溶性重組蛋白質。所有重組受體.Fc融合蛋白質藉由蛋白質A瓊脂糖(Sepharose)珠粒(Pharmacia)純化並用0.1M甘胺酸-HCl(pH0.3)溶析。Dengue fever (DV) infection of DC-SIGN media human dendritic cells is known (Tassaneetrithepet al., J Exp Med, 2003. 197(7): p. 823-9). To understand the cause of DV, determine whether DV is associated with other membrane-bound C-type lectin receptors and C-like forms from dendritic cells, macrophages, natural killer cells, and peripheral blood mononuclear cells (PBMCs). Lectin receptor interaction is important. To achieve this, the extracellular region of DVLR1/CLEC5A (MDL-1), dendritic cell lectin-1, KCR, and DC-SIGN (as a positive control block) was fused to the Fc portion of human IgG1. More specifically, primers of DC-SIGN (SEQ ID NO: 17 and SEQ ID NO: 18), primers of DVLR1/CLEC5A (SEQ ID NO: 21 and SEQ ID NO: 22), and primers of dendritic cell lectin-1 were used ( SEQ ID NO: 25 and SEQ ID NO: 26) and the introduction of KCR (forward: 5'-CAGCCTTGGAGACCTGAGT-3' SEQ ID NO: 37; reverse 5'-TAGCCTACTCTGGCCGC-3' SEQ ID NO: 38) to generate amplified cDNA fragments . Each forward primer has an additional BamH1 site, and each reverse primer has an additional EcoRI site to accelerate the subcloning of the amplified cDNA into the pCDNA3.1 (Invitrogen) mammalian expression vector containing the human IgG1 Fc portion. The resulting vector was then transfected into 293 FreeStyle cells (lnvitrogen) to produce soluble recombinant proteins. All recombinant receptor.Fc fusion proteins were purified by Protein A Sepharose beads (Pharmacia) and eluted with 0.1 M glycine-HCl (pH 0.3).

將1μg各受體.Fc融合蛋白質塗佈在微量滴定平皿上並置於4℃整夜。然後將在結合緩衝液(1%BSA,2 mM CaCl₂ ,2 mM MgCl₂ ,50 mM Tris-HCl pH 7.5,150 mM NaCl)中之DV病毒株16681(DEN2系)(5x10⁶ 個粒子)加至平皿中以及將該平皿培育2小時。沖洗除去未結合之病毒後，施用生物素化抗-DEN2套膜蛋白質抗體(Wu et al.,J Virol,2002.76(8)：p.3596-604)，使其與病毒進行結合1小時。然後將經稀釋之綴合有辣根過氧化酶之鏈酶抗生物素蛋白加至平皿中，繼而進行1小時之培育。然後加入TMB受質並將平皿用ELISA讀數器在OD450 nm測量。1 μg of each receptor.Fc fusion protein was plated on a microtiter plate and placed at 4 ° C overnight. Then add DV strain 16681 (DEN2 line) (5x10 ⁶ particles) in binding buffer (1% BSA, 2 mM CaCl ₂ , 2 mM MgCl ₂ , 50 mM Tris-HCl pH 7.5, 150 mM NaCl). The plate was incubated for 2 hours in the dish. After washing to remove unbound virus, biotinylated anti-DEN2 envelope protein antibody (Wu et al., J Virol, 2002. 76(8): p. 3596-604) was administered and allowed to bind to the virus for 1 hour. The diluted streptavidin conjugated with horseradish peroxidase was then added to the plate and incubated for 1 hour. TMB substrate was then added and the plates were measured at OD450 nm using an ELISA reader.

結果示於圖8A中(**表示p<0.01；***表示p<0.001(學生t試驗))。結果顯示除了DC-SIGN(陽性對照組)之外，DV亦與DVLR1/CLEC5A結合。為了確證該結果，用人類IgG1(陰性對照組)、DC-SIGN.Fc、KCR.Fc及DVLR1.Fc進行免疫沉澱研究。更特定而言，將5x10⁶ 個登革熱病毒粒子與5 μg之各蛋白質一起培育，然後加入蛋白質A珠粒。將生成之免疫複合物沖洗，藉由SDS-PAGE分離並移至硝基纖維素膜上。然後將該膜用生物素化之抗-DEN2套膜蛋白質抗體偵測並用綴合有辣根過氧化酶之鏈黴抗生物素蛋白顯色。此等結果示於圖8B中。此等結果顯示只有DC-SIGN.Fc即及DVLR1/CLEC5A.Fc能免疫沉澱登革熱病毒。The results are shown in Fig. 8A (** indicates p <0.01; *** indicates p < 0.001 (student t test)). The results showed that in addition to DC-SIGN (positive control group), DV also binds to DVLR1/CLEC5A. To confirm the results, immunoprecipitation studies were performed with human IgG1 (negative control), DC-SIGN.Fc, KCR.Fc, and DVLR1.Fc. More particularly, the dengue incubated with 5x10 ⁶ virus particles and 5 μg of each protein, and Protein A beads were added. The resulting immune complex was washed, separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then detected with a biotinylated anti-DEN2 envelope protein antibody and developed with streptavidin conjugated with horseradish peroxidase. These results are shown in Figure 8B. These results show that only DC-SIGN.Fc and DVLR1/CLEC5A.Fc can immunoprecipitate dengue virus.

於存在能螯合Ca⁺⁺ 陽離子之EDTA(10 mM)下重覆進行微量滴定平皿之檢定分析。此等結果(圖8C)顯示與登革熱病毒之DVLR1/CLEC5A結合為Ca⁺⁺ 非依存性，而DC-SIGN結合為Ca⁺⁺ 依存性(***表示p<0.001，學生t試驗)。Characterization of microtiter plates was repeated in the presence of EDTA (10 mM) capable of chelation of Ca ⁺⁺ cations. These results (Fig. 8C) show that binding to DVLR1/CLEC5A of dengue virus is Ca ^++- independent, while DC-SIGN binding is Ca ^++- dependent (*** indicates p<0.001, Student t test).

亦反覆進行微量滴定平皿檢定分析，以測定DVLR1/CLEC5A.Fc融合蛋白質與DV粒子(5x10⁶ )之結合，其中該DV粒子曾接受下列處理：1)與500U糖苷酶PNGaseF(New England Biolabs,Inc.)於37℃預處理整夜；或2)用二硫蘇糖醇(DTT)(0.1M)處理；或3)於95℃預培育5分鐘；或4)進行UV照射5分鐘。結果顯示於圖8D中(星號表示DVLR1/CLEC5A.Fc融合蛋白質之結合親和力藉由病毒之修改，而與未經處理之病毒相較發生改變)；**p<0.01，***p<0.001，學生t試驗)。結果顯示DV用PNGaseF預處理會顯著抑制與DVLR1/CLEC5A.Fc之交互作用，以及用熱或二硫蘇糖醇之任一者預處理幾乎會完全抑制DVLR1/CLEC5A.Fc結合，但不會抑制DC-SIGN.Fc與DV之結合。此暗示DV之糖抗原決定部位及三次元組態對於與DVLR1/CLEC5A之結合頗為重要。A microtiter plate assay was also performed to determine the binding of the DVLR1/CLEC5A.Fc fusion protein to DV particles (5x10 ⁶ ), which had been subjected to the following treatments: 1) with 500 U glycosidase PNGaseF (New England Biolabs, Inc .) pretreatment overnight at 37 ° C; or 2) treatment with dithiothreitol (DTT) (0.1 M); or 3) pre-incubation at 95 ° C for 5 minutes; or 4) UV irradiation for 5 minutes. The results are shown in Figure 8D (the asterisk indicates that the binding affinity of the DVLR1/CLEC5A.Fc fusion protein is altered by the virus compared to the untreated virus); **p<0.01, ***p<0.001 , student t test). The results showed that DV pretreatment with PNGaseF significantly inhibited the interaction with DVLR1/CLEC5A.Fc, and pretreatment with either heat or dithiothreitol almost completely inhibited DVLR1/CLEC5A.Fc binding, but did not inhibit The combination of DC-SIGN.Fc and DV. This suggests that the sugar epitope and the three-dimensional configuration of DV are important for binding to DVLR1/CLEC5A.

為了評估在免疫細胞上DVLR1/CLEC5A之表現，對於人類多核(PMN)細胞(嗜中性球)、PBMCs、巨嗜細胞及樹狀細胞進行流動式細胞計數。PMNs及PBMCs分別藉由先前所述之聚葡萄糖(dextran)沉降(Kuan et al.,Br.J.Pharmacol.,2005,145(4)：460-468)並用Ficoll-Paque(Amersham Biosciences,Piscataway,NJ)進行標準密度梯度離心而自健康人類捐血者之全血單離。將純化嗜中性球再懸浮於磷酸鹽水緩衝液(PBS,pH 7.4)中，以使紅血球低張溶裂。繼而藉由採用抗-CD14微珠(Miltenyi Biotec GmbH,Bergisch Gladbach,Germany)之VARIOMACS技術進行高梯度磁性篩選，以從PBMCs純化CD14+細胞，然後在添加有10 ng/ml人類M-CSF(R&D Systems,Minneapolis,MN)之完全RPMI-1640培養基(Life Technologies,Gaithersburg,MD)中培養6日(Chang et al.,J.Leukoc Biol,2004,75(3)：486-494)。藉由在添加10%牛胎血清、800 U/ml人類GM-CSF(Leucomax；Schering-Plough,Kenilworth,NJ)及500 U/ml人類IL-4(R&D Systems)之RPMI 1640培養基中培養6日，從吸附之PBMCs產生樹狀細胞(DC)(不成熟DCs)。為了製備成熟的經活化DCs，將不成熟的DCs與經γ射線照射(5500 rad)之CD40配體(CD40L)-表現性L細胞(DNAX Research Institute,Palo Alto,CA)以3：1之比率一起培育36小時(Hsu et al.,J Immunol.,2002,168(10)：4846-4853)。To assess the performance of DVLR1/CLEC5A on immune cells, flow cytometry was performed on human multinuclear (PMN) cells (neutrophils), PBMCs, macrophages, and dendritic cells. PMNs and PBMCs were separately sedimented by dextran as previously described (Kuan et al., Br. J. Pharmacol., 2005, 145(4): 460-468) and Ficoll-Paque (Amersham Biosciences, Piscataway, NJ) Performs standard density gradient centrifugation and separates whole blood from healthy human donors. The purified neutrophil was resuspended in phosphate water buffer (PBS, pH 7.4) to cause red blood cells to be lytic. High-gradient magnetic screening was then performed by using the VARIOMACS technique of anti-CD14 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) to purify CD14+ cells from PBMCs, followed by the addition of 10 ng/ml human M-CSF (R&D Systems Cultured in complete RPMI-1640 medium (Life Technologies, Gaithersburg, MD) of Minneapolis, MN) (Chang et al., J. Leukoc Biol, 2004, 75(3): 486-494). Incubation for 6 days in RPMI 1640 medium supplemented with 10% fetal calf serum, 800 U/ml human GM-CSF (Leucomax; Schering-Plough, Kenilworth, NJ) and 500 U/ml human IL-4 (R&D Systems) Dendritic cells (DC) (immature DCs) are produced from adsorbed PBMCs. To prepare mature activated DCs, the ratio of immature DCs to gamma-irradiated (5500 rad) CD40 ligand (CD40L)-expressing L cells (DNAX Research Institute, Palo Alto, CA) at a ratio of 3:1 Incubate for 36 hours together (Hsu et al., J Immunol., 2002, 168(10): 4846-4853).

對於上述細胞類型進行流動式細胞計數，其中使用綴合有FITC之抗-DVLR1/CLEC5A單株抗體(R&D Systems,Minneapolis,MN)或綴合有FITC之抗-DC-SIGN單株抗體(ED PharMingen)，連同綴合有藻紅素(PE)之抗CD3、CD19、CD56、CD14及CD66抗體，以進行雙重染色(BD PharMingen)。亦對相配之同種型(isotype)對照組(就DVLRI mAb而言為IgG2b；就DC-SIGN而言為IgG 1；Sigma)進行該表面染色，以提供背景值資料。藉由FACSCalibur流動式細胞計數器(Becton Dickinson)及CellQuest軟體(Becton Dickinson)來分析螢光，選通(gate)CD標記陽性細胞以測定DVLR1/CLEC5A或DC-SIGN之表現。該等結果示於圖9A(DVLR/CLEC5A)及圖9B(DC-SIGN)(陰影區域代表同種型對照組)。此等結果表示DC-SIGN主要在不成熟樹狀細胞上表現以及在巨噬細胞上微弱表現。該等結果亦顯示DVLR1/CLEC5A在CD14+衍化之巨噬細胞(MΦ)、CD66+ PMNs及剛單離之CD14+ PBMCs之表面上檢測到，但未在CD14+衍化的不成熟及成熟樹狀細胞上檢測到。此與先前所觀察到之「DVLR1/CLEC5A mRNA在人類單核球及巨噬細胞中表現，但不在樹狀細胞中表現」一致(Bakker et al.,Proc.Natl.Acad Sci USA,1999,96(17)：9792-9796)。Flow cytometry was performed for the above cell types using anti-DVLR1/CLEC5A monoclonal antibody conjugated with FITC (R&D Systems, Minneapolis, MN) or anti-DC-SIGN monoclonal antibody conjugated with FITC (ED PharMingen) ), together with anti-CD3, CD19, CD56, CD14 and CD66 antibodies conjugated with phycoerythrin (PE) for double staining (BD PharMingen). This surface staining was also performed on a matched isotype control group (IgG2b for DVLRI mAb; IgG 1 for Sigma 1; Sigma) to provide background value data. Fluorescence was analyzed by FACSCalibur flow cytometer (Becton Dickinson) and CellQuest software (Becton Dickinson), and CD-labeled positive cells were gated to determine the performance of DVLR1/CLEC5A or DC-SIGN. The results are shown in Figure 9A (DVLR/CLEC5A) and Figure 9B (DC-SIGN) (shaded areas represent isotype control groups). These results indicate that DC-SIGN is mainly expressed on immature dendritic cells and weakly expressed on macrophages. These results also showed that DVLR1/CLEC5A was detected on the surface of CD14+-derived macrophages (MΦ), CD66+ PMNs, and CD14+ PBMCs, but not on CD14+-derived immature and mature dendritic cells. . This is consistent with the previously observed "DVLR1/CLEC5A mRNA is expressed in human monocytes and macrophages but not in dendritic cells" (Bakker et al., Proc. Natl. Acad Sci USA, 1999, 96). (17): 9792-9796).

在該實施例中呈現之結果顯示本文所揭示之以受體.Fc融合蛋白質為主體之方法可用於測定能與特異性病原體諸如登革熱病毒結合之先天性免疫受體之種類。其最終允許鑑定可與病原體交互作用之細胞類型，並進一步提供治療或預防病原體感染之新穎標的。舉例言之，本文所揭示之結果暗示防止DV與DVLR1/CLEC5A結合之藥劑可用於預防或治療目的。舉例言之，對抗DVLR1/CLEC5A之單株抗體可由熟悉本技術人士製造，以防止DV與DVLR1/CLEC5A結合。再者，既然DV為黃病毒科家族之成員，該結果暗示DVLR1/CLEC5A可與在相同家族中之其他病毒交互作用，該等病毒例如為黃熱病毒屬中之病毒(諸如西尼羅熱病毒、日本腦脊髓炎病毒(JEV)、黃熱病毒及蜱傳腦脊髓炎病毒)或肝炎病毒屬中之病毒(諸如C型肝炎病毒)。所以DVLR1/CLEC5A亦可做為此等病毒之治療或預防標的。此外，既然DVLR1/CLEC5A為模式識別受體，DVLR1/CLEC5A可做為其他具套膜病毒之治療或預防標靶。The results presented in this example show that the method disclosed herein, which is based on the receptor.Fc fusion protein, can be used to determine the class of innate immune receptors that bind to specific pathogens such as dengue virus. It ultimately allows identification of cell types that can interact with pathogens and further provides novel targets for treating or preventing infection by pathogens. For example, the results disclosed herein suggest that agents that prevent DV binding to DVLR1/CLEC5A can be used for prophylactic or therapeutic purposes. For example, monoclonal antibodies against DVLR1/CLEC5A can be made by those skilled in the art to prevent binding of DV to DVLR1/CLEC5A. Furthermore, since DV is a member of the Flaviviridae family, this result suggests that DVLR1/CLEC5A can interact with other viruses in the same family, such as viruses in the genus Feverage (such as West Nile Virus) , Japanese encephalomyelitis virus (JEV), yellow fever virus and sputum encephalomyelitis virus) or viruses in the genus Hepatitis virus (such as hepatitis C virus). Therefore, DVLR1/CLEC5A can also be used as a treatment or prevention target for such viruses. In addition, since DVLR1/CLEC5A is a pattern recognition receptor, DVLR1/CLEC5A can be used as a therapeutic or preventive target for other enveloped viruses.

實施例12：登革熱病毒誘導之DAP12磷醯化由Example 12: Dengue virus-induced DAP12 phosphorylation by DVLR1/CLEC5A媒介DVLR1/CLEC5A medium

DVLR1/CLEC5A(MDL-1)為包含187個胺基酸長之第II型穿膜蛋白質，其包括在穿膜區能使其與DAP12(具12 kDa之DNAX活化蛋白質)配對之帶電殘基(Bakker et al.,Proc.Natl.Acad Sci USA,1999,96(17)：9792-9796)。DAP12為以二硫鍵鍵結之同元二聚穿膜蛋白質，其具有極小的細胞外區域，在穿膜區域具有帶電的天冬胺酸以及在其細胞質尾部具有ITAM(免疫受體酪胺酸系活化基元)。由於在CD14+巨噬細胞上DV與DVLR1/CLEC5A結合且由於DAP12具有ITAM，因此引發測定在CD14+巨噬細胞中DV是否會誘生DAP12磷醯化之興趣。所以，在使用Chen等人(J.Virol.2002,76(19)：9877-9887)所揭示之方法並經略微修改下，將CD14+巨噬細胞以DV感染。簡言之，將終末分化之巨噬細胞用不完全的RPMI培養基沖洗一次，以移除培養基中之牛胎血清。然後將細胞用DV以不同的感染重複數(MOI)感染。將該等病毒與細胞在不含血清之RPMI中於37℃培育2.5小時，以允許病毒吸附。將培養平皿每30分鐘溫和攪動1次，以達成最佳的病毒-細胞接觸。之後，未吸收的病毒藉由用不含血清之RPMI沖洗細胞單層2次，然後進行培育1次而移除，分別收取不含細胞之上清液並分成數份貯存於-80℃，直至進行感染病毒製造及細胞激素分泌之檢定分析(見實施例13)。感染病毒滴度藉由在BHK-21細胞上之成斑檢定分析而測定。於結晶紫覆蓋後7日，藉由目視檢查計算斑數目，以測定每毫升上清液中之成斑單位(PFU)之數目(Lin et al.,J.Virol.,1998,72(12)：9729-9737)。為了檢測細胞內之DV抗原，將經感染之細胞用1%三聚甲醛固定並用0.1%皂素可通透化，繼而用NS3 mAb(Lin et al.,J.Virol.,1998,72(12)：9729-9737)或相配之同種型對照組(IgG1；Sigma)染色。培育1小時後，加入綴合有PE之山羊F(ab)'抗-鼠IgG二次抗體以進行螢光檢測，並藉由FACSCalibur流動式細胞計數器及CellQuest軟體來分析螢光。DVLR1/CLEC5A (MDL-1) is a type II transmembrane protein comprising 187 amino acids long comprising a charged residue capable of pairing it with DAP12 (a 12 kDa DNAX activating protein) in the transmembrane region ( Bakker et al., Proc. Natl. Acad Sci USA, 1999, 96(17): 9792-9796). DAP12 is a disulfide-bonded homodimeric penetrating membrane protein with a very small extracellular region, charged aspartic acid in the transmembrane region, and ITAM (immunoreceptor tyrosine) at its cytoplasmic tail. System activation unit). Since DV binds to DVLR1/CLEC5A on CD14+ macrophages and because DAP12 has ITAM, it elicits an interest in determining whether DV induces DAP12 phosphorylation in CD14+ macrophages. Therefore, CD14+ macrophages were infected with DV using the method disclosed by Chen et al. (J. Virol. 2002, 76(19): 9787-9887) with slight modifications. Briefly, terminally differentiated macrophages were washed once with incomplete RPMI medium to remove bovine fetal serum from the culture medium. Cells were then infected with DV at different infection repeat numbers (MOI). The viruses were incubated with cells in serum-free RPMI for 2.5 hours at 37 °C to allow for virus adsorption. The culture plates were gently agitated once every 30 minutes to achieve optimal virus-cell contact. Thereafter, the unabsorbed virus was removed by washing the cell monolayer twice with serum-free RPMI and then culturing once, and the supernatant-free supernatant was separately collected and stored in several portions at -80 ° C until Verification analysis of infection virus production and cytokine secretion was performed (see Example 13). Infectious virus titers were determined by plaque assay on BHK-21 cells. The number of plaques per ml of supernatant was determined by visual inspection on the 7th day after crystal violet coverage (Lin et al., J. Virol., 1998, 72(12). :9729-9737). To detect DV antigens in cells, infected cells were fixed with 1% paraformaldehyde and permeabilized with 0.1% saponin, followed by NS3 mAb (Lin et al., J. Virol., 1998, 72 (12). ): 9729-9737) or matched isotype control (IgG1; Sigma) staining. After 1 hour of incubation, goat F(ab)' anti-mouse IgG secondary antibody conjugated with PE was added for fluorescence detection, and fluorescence was analyzed by a FACSCalibur flow cytometer and CellQuest software.

結果示於圖10A-D中。於以MOI=5感染後48小時，在巨噬細胞之細胞溶質中DV非結構性蛋白質3(NS3)藉由流動式細胞計數法檢測(圖10A；灰色組織圖為同種型抗體對照組)。細胞外病毒滴度於感染後各種時間測量，且其顯示當巨噬細胞以活DV感染時，病毒粒子釋出至培養上清液，但以經UV-照射之DV(UV-DV；於相距5至10cm處，在冰上接受254 nm輻射照射15分鐘)感染時則否(圖10B)。The results are shown in Figures 10A-D. At 48 hours post infection with MOI=5, DV non-structural protein 3 (NS3) was detected by flow cytometry in the cytosol of macrophages (Fig. 10A; gray tissue map is an isotype antibody control group). The extracellular virus titer was measured at various times after infection, and it was shown that when macrophages were infected with live DV, the virions were released to the culture supernatant, but with UV-irradiated DV (UV-DV; 5 to 10 cm, 254 nm radiation exposure on ice for 15 minutes) No infection (Fig. 10B).

以各種MOIs感染後2小時(MOI=0.05-30，感染後2小時)，以及以固定MOI(MOI=5)感染一段時程(感染後2至48小時)後，研究DAP12之磷醯化。特定而言，為了檢測磷-DAP12，將巨噬細胞用DV以適當的MOI刺激一段適當的時間，然後在溶裂緩衝液(50mM Tris-HCI[pH7.5]、150mM NaCI、1% Triton X-100、0.1% SDS、5mM EDTA、10mM NaF、1mM原釩酸鈉及蛋白酶抑制劑混合錠[Roche])中進行溶裂。將等量的全細胞萃取物用DAP12兔多株抗體(Santa Cruz Biotechnology Inc,CA)及蛋白質A瓊脂糖(Sepharose)(Amersham Biosciences AB)於4℃進行免疫沉澱4小時。培育後，將免疫複合物沖洗3次以及藉由SDS-PAGE分離，繼而移至硝基纖維素膜及用抗-磷醯酪胺酸抗體(4G10；Upstate Biotechnology,Inc)偵測。將免疫轉漬片用綴合有HRP之二次抗體及增強之化學發光(Amersham)顯色。為了進行再偵測，將膜用強再偵測套組剝除並用DAP12抗體轉漬。Phosphorylation of DAP12 was studied 2 hours after infection with various MOIs (MOI = 0.05-30, 2 hours post infection) and with a fixed MOI (MOI = 5) for a period of time (2 to 48 hours after infection). Specifically, to detect phospho-DAP12, macrophages were stimulated with DV at the appropriate MOI for a suitable period of time, then in a lysis buffer (50 mM Tris-HCI [pH 7.5], 150 mM NaCI, 1% Triton X). Dissolution was carried out in -100, 0.1% SDS, 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate and protease inhibitor mixed ingot [Roche]. Equal amounts of whole cell extracts were immunoprecipitated with DAP12 rabbit polyclonal antibody (Santa Cruz Biotechnology Inc, CA) and Protein A Sepharose (Amersham Biosciences AB) for 4 hours at 4 °C. After incubation, the immune complexes were washed 3 times and separated by SDS-PAGE, then transferred to a nitrocellulose membrane and detected with an anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Inc.). The immunostained tablets were developed with secondary antibodies conjugated with HRP and enhanced chemiluminescence (Amersham). For re-detection, the membrane was stripped with a strong re-detection kit and transfected with DAP12 antibody.

將在各種MOIs下得到之結果示於圖10C中，而時程實驗結果示於圖10D中。此等結果顯示在DV感染後2小時，DAP12磷醯化強度之增加自MOI=0.05開始升高，在MOI=5時到達尖峯值(圖10C)。於DV感染後2小時檢測DAP12磷醯化，於12小時達到峯值，並持續至少48小時(圖10D)。縱使UV-DV在CD14+巨噬細胞中無法複製且在斑檢定分析中顯示沒有活性(圖10B)，DAP12於2小時亦被磷醯化，且磷醯化之DAP12於12小時仍可檢測得到，惟其強度遠比活DV所誘生者弱許多(圖10D；UV-DV)。此暗示DV所誘生之DAP12磷醯化具有二期：第I期(最初6小時)為複製-非依存性，而第II期(12小時後)為複製-依存性。The results obtained under various MOIs are shown in Fig. 10C, and the results of time course experiments are shown in Fig. 10D. These results show that the increase in DAP12 phosphorylation intensity increased from MOI = 0.05 at 2 hours after DV infection and reached a peak at MOI = 5 (Fig. 10C). DAP12 phosphonium was detected 2 hours after DV infection and peaked at 12 hours for at least 48 hours (Figure 10D). Even though UV-DV was unable to replicate in CD14+ macrophages and showed no activity in plaque assays (Fig. 10B), DAP12 was also phosphorylated at 2 hours, and phosphonated DAP12 was still detectable at 12 hours. However, its intensity is much weaker than that induced by live DV (Fig. 10D; UV-DV). This suggests that the DAP12 phosphonium induced by DV has two phases: phase I (first 6 hours) is replication-non-dependency, and phase II (after 12 hours) is replication-dependency.

為了確證DAP12磷醯化係經由DVLR1/CLEC5A，用短髮夾狀RNA(shRNA)進行之RNA干擾(RNAi)抑制在CD14+巨噬細胞中DVLR1/CLEC5A之表現並如上述檢定分析DAP12之磷醯化。特定而言，人類DVLR1/CLEC5A之編碼區用下列DVLR1/CLEC5A siRNA定標：5'-TTGTTGGAATGACCTTAT-3'To confirm that DAP12 phosphonium is expressed by DVLR1/CLEC5A, RNA interference (RNAi) with short hairpin RNA (shRNA) inhibits the expression of DVLR1/CLEC5A in CD14+ macrophages and analyzes the phosphorylation of DAP12 as described above. . In particular, the coding region of human DVLR1/CLEC5A was calibrated with the following DVLR1/CLEC5A siRNA: 5'-TTGTTGGAATGACCTTAT-3'

序列編號：39Serial number: 39

此段係用得自Brummelkamp et al.,Science,2002,296(5567)：550-553之環形序列(TTCAAGAGA)修改以建立shRNA。此處所使用之聚合酶III終止子段為TTTTTT。將shRNA選殖入pLL3.7基因沉默載體(Rubinson et al.,Nat.Genet.,2003,33(3)：401-406)，該載體含有loxP部位、驅動增強之綠色螢光蛋白質(EGFP)之表現之CMV(巨細胞病毒)啟動子以及具有下游限制部位(HpaI及XhoI)之U6啟動子。DC-SIGN shRNA構築體亦藉由將構築體pSUPER-siDC-SIGN(Tassaneetrithep et al.,如同上文)所含之shRNA次選殖入經HpaI/XhoI消化之pLL3.7載體而構築。依照製造商之說明書，將構築體用Amaxa套組(Gaithersburg,MD)電穿孔入巨噬細胞中。簡言之，將巨噬細胞(6x 10⁶ )如上述收取並再懸浮於100 μL之核因子溶液。加入siRNA(5μg)或載體對照組後，將細胞用Amaxa程式Y-001電穿孔並經16小時讓其復原。藉由分別使用抗-DVLR1/CLEC5A及DC-SIGN單株抗體(R&D Systems)進行免疫轉漬而轉染後24小時，分析DVLR1及DC-SIGN沉默之效率。This was modified with the circular sequence (TTCAAGAGA) from Brummelkamp et al., Science, 2002, 296 (5567): 550-553 to establish shRNA. The polymerase III terminator segment used herein is TTTTTT. shRNA was cloned into the pLL3.7 gene silencing vector (Rubinson et al., Nat. Genet., 2003, 33(3): 401-406), which contains a loxP site, driving enhanced green fluorescent protein (EGFP) The CMV (cytomegalovirus) promoter and the U6 promoter with downstream restriction sites (HpaI and XhoI). The DC-SIGN shRNA construct was also constructed by subcloning the shRNA contained in the construct pSUPER-siDC-SIGN (Tassaneetrithep et al., supra) into the HLAI/XhoI digested pLL3.7 vector. The constructs were electroporated into macrophages using the Amaxa kit (Gaithersburg, MD) according to the manufacturer's instructions. Briefly, macrophages ( ⁶ x 106) were harvested and resuspended in 100 μL of nuclear factor solution as described above. After addition of siRNA (5 μg) or vehicle control, cells were electroporated with Amaxa program Y-001 and allowed to recover over 16 hours. The efficiency of DVLR1 and DC-SIGN silencing was analyzed by immunoblotting using anti-DVLR1/CLEC5A and DC-SIGN monoclonal antibodies (R&D Systems), respectively, 24 hours after transfection.

結果示於圖11中。藉由電穿孔導入對照載體pLL3.7或DC-SIGN-shRNA之CD14+巨噬細胞以DV感染後，未顯示DAP12磷醯化降低。相對照地，在DV感染之前先藉由電穿孔導入DVLR1/CLEC5A-shRNA之CD14+巨噬細胞中，DAP12磷醯化急遽降低。所以，可以獲得下述結論：DV-誘導之DAP12磷醯化係經由DVLR1而發生。The results are shown in FIG. The D1412 phosphorylation was not shown to be reduced after infection with DV by electroporation of CD14+ macrophages introduced into the control vector pLL3.7 or DC-SIGN-shRNA. In contrast, DAP12 phosphorylation was rapidly reduced by electroporation of CD14+ macrophages introduced into DVLR1/CLEC5A-shRNA prior to DV infection. Therefore, it can be concluded that DV-induced DAP12 phosphorylation occurs via DVLR1.

實施例13:DVLR1/CLEC5A涉及DV-媒介之TNF-α釋出，但不涉及進入CD14+巨噬細胞Example 13: DVLR1/CLEC5A is involved in DV-mediated TNF-α release, but does not involve entry into CD14+ macrophages

於DV感染時，CD14+巨噬細胞分泌促發炎性細胞激素及化學激素，包括腫瘤壞死因子-α(TNF-α)、α-干擾素(IFN-α)、MIP-1α及IL-8(Chen et al,如同上文)。使用市售ELISA套組測量經DV-感染之CD14+巨噬細胞之培養上清液中之TNF-α濃度。針對活DV及UV-DV二者，於不同MOIs及於感染後不同時間進行測量。結果示於圖12A-C(誤差長條圖代表與複製三份之平均值之標準誤差，而星號表示細胞激素產量之統計學上差異值，*=p<0.05；**=p<0.01；***=p<0.001)。此等結果顯示感染後6小時，活DV及DV-UV二者在0.05-30之MOI範圍內對於TNF-α分泌具有相似的作用(圖12A)。於感染後12小時，只有活DV之TNF-α分泌以劑量依存(增加MOI)方式增加。就感染後12小時之感染有UV-DV之細胞而言，TNF-α濃度在所有MOIs均保持相同(圖12B)。圖12C顯示TNF隨著時程之測量值。此等值顯示當用活DV以MOI=5感染時，TNF-α分泌量由於6小時之8 pg/ml迅速增至於12小時之85 pg/ml，並在48小時達到高峰(350 pg/ml)。不過，當與UV-DV一起培育時，TNF-α分泌從8 pg/ml(於6小時)降至5 pg/ml(於12小時)。此暗示初始反應(於6小時)與病毒複製無關，而TNF-α分泌之較後期(12小時後)與DV複製有關。When infected with DV, CD14+ macrophages secrete inflammatory cytokines and chemical hormones, including tumor necrosis factor-α (TNF-α), α-interferon (IFN-α), MIP-1α and IL-8 (Chen Et al, as above). The concentration of TNF-α in the culture supernatant of DV-infected CD14+ macrophages was measured using a commercially available ELISA kit. For both live DV and UV-DV, measurements were made at different MOIs and at different times after infection. The results are shown in Figures 12A-C (the error bars represent the standard error of the mean of the replicates, and the asterisks indicate the statistical difference in cytokine production, * = p < 0.05; ** = p < 0.01; ***=p<0.001). These results show that both live DV and DV-UV have similar effects on TNF-[alpha] secretion in the MOI range of 0.05-30 at 6 hours post infection (Fig. 12A). At 12 hours post infection, only TNF-[alpha] secretion of live DV increased in a dose dependent (increased MOI) manner. The TNF-α concentration remained the same in all MOIs for the cells infected with UV-DV 12 hours after infection (Fig. 12B). Figure 12C shows the measured values of TNF over time. This value shows that when infected with live DV at MOI=5, the amount of TNF-α secreted rapidly increased from 8 pg/ml at 6 hours to 85 pg/ml at 12 hours and peaked at 48 hours (350 pg/ml). ). However, when incubated with UV-DV, TNF-[alpha] secretion decreased from 8 pg/ml (at 6 hours) to 5 pg/ml (at 12 hours). This suggests that the initial response (at 6 hours) is not associated with viral replication, whereas the later phase of TNF-[alpha] secretion (after 12 hours) is associated with DV replication.

先前已證明DC-SIGN會與DV交互作用，以媒介病毒進入樹狀細胞。使用先前實施例之RNAi方法及試劑，研究在經DV-感染之CD14+巨噬細胞中DC-SIGN-shRNA及DVLR1/CLEC5A-shRNA對於NS3表現之作用。圖13A顯示DC-SIGN-shRNA及DVLR1/CLEC5A-shRNA可分別使其對應蛋白質表現減少(knock down)(pWTSI及pLL3.7為無插子對照組)。圖13B圖示流動式細胞計數分析之結果並例示說明只有DC-SIGN-shRNA能使CD14+巨噬細胞中之DVNS3表現減少。該結果藉由使用抗-DS3抗體進行免疫螢光共軛焦顯微鏡檢而確證。圖13C例示說明在藉由電穿孔導入shANA構築體之細胞之上清液中，病毒滴度之即時PCR分析。該等結果表示只有DC-SIGN-shRNA能減少經DV感染之細胞之上清液中之病毒滴度。DC-SIGN has previously been shown to interact with DV to mediate viruses into dendritic cells. The effect of DC-SIGN-shRNA and DVLR1/CLEC5A-shRNA on NS3 expression in DV-infected CD14+ macrophages was investigated using the RNAi methods and reagents of the previous examples. Figure 13A shows that DC-SIGN-shRNA and DVLR1/CLEC5A-shRNA can knock down their corresponding protein performance (pWTSI and pLL3.7 are no-insertion control groups). Figure 13B illustrates the results of flow cytometric analysis and exemplifies that only DC-SIGN-shRNA can reduce DVNS3 expression in CD14+ macrophages. The results were confirmed by immunofluorescence conjugated focus microscopy using an anti-DS3 antibody. Figure 13C illustrates an instant PCR analysis of viral titers in supernatants of cells introduced into shANA constructs by electroporation. These results indicate that only DC-SIGN-shRNA can reduce the virus titer in the supernatant above the DV-infected cells.

實施例14:DVLR1/CLEC5A涉及DV所誘導之促發炎性細胞激素從CD14+巨噬細胞之釋出Example 14: DVLR1/CLEC5A is involved in the release of proinflammatory cytokines induced by DV from CD14+ macrophages

就以DV(MOI=5)感染之CD14+巨噬細胞而言，於依照先前實施例之方法(2.5小時轉染)使DVLR1/CLEC5A及DC-SIGN基因表現下調(knock down)後，使用ELISA評估細胞激素釋出模式。在最初12小時，DC-SIGN-shRNA不會影響TNF-α、MIP-1α、IFN-α、IL-6或IL-8之分泌。參見圖14A-B(誤差長條圖代表與複製三份之平均值之標準誤差，而星號表示與對照實驗相較，在統計學上有顯著之差異；*=p<0.05；**=p<0.01；***=p<0.001)。48小時後，DC-SIGN-shRNA對於TNF-α、MIP-1α、IFN-α及IL-6分泌具有輕微的抑制作用(少於20%)；IL-8分泌則不受影響。既然DC-SIGN參與病毒進入及複製，該觀察暗示初始細胞激素分泌(最初12小時)與DV複製無關。相對地，DVLR1/CLEC5A基因表現下調急遽地壓制(p<0.005)TNF-α、MIP-1α、IFN-α及IL-8之分泌，但不會壓制IL-6之分泌。此暗示DVLR1/CLEC5A主導DV所誘導之細胞激素來自CD14+巨噬細胞之釋放。所以防止DV與DVLR1/CLEC5A結合之治療劑可用於在人類中防止DV所誘導之細胞激素釋放之不良作用。舉例言之，防止DVLR1/CLEC5A與DV交互作用之單株抗體將可用於預防或治療DV所誘導之登革熱休克症候群(DSS)或出血性登革熱(DHF)。For CD14+ macrophages infected with DV (MOI=5), the DVLR1/CLEC5A and DC-SIGN genes were down-regulated after the method according to the previous example (2.5 hour transfection), and evaluated by ELISA. Cytokine release pattern. At the first 12 hours, DC-SIGN-shRNA did not affect the secretion of TNF-α, MIP-1α, IFN-α, IL-6 or IL-8. See Figures 14A-B (the error bar graph represents the standard error of the mean of the replicates and the asterisk indicates a statistically significant difference compared to the control experiment; *=p<0.05;**=p <0.01; ***=p<0.001). After 48 hours, DC-SIGN-shRNA had a slight inhibitory effect (less than 20%) on TNF-α, MIP-1α, IFN-α and IL-6 secretion; IL-8 secretion was not affected. Since DC-SIGN is involved in viral entry and replication, this observation suggests that initial cytokine secretion (first 12 hours) is not associated with DV replication. In contrast, the DVLR1/CLEC5A gene was down-regulated to rapidly suppress (p<0.005) secretion of TNF-α, MIP-1α, IFN-α, and IL-8, but did not suppress IL-6 secretion. This suggests that the cytokine induced by DVLR1/CLEC5A-dominant DV is derived from the release of CD14+ macrophages. Therefore, a therapeutic agent that prevents DV from binding to DVLR1/CLEC5A can be used to prevent the adverse effects of DV-induced cytokine release in humans. For example, monoclonal antibodies that prevent DVLR1/CLEC5A from interacting with DV will be useful in the prevention or treatment of DV-induced dengue shock syndrome (DSS) or hemorrhagic dengue fever (DHF).

實施例15：拮抗性抗-DVLR1/CLEC5A單株抗體(mAbS)遏止因登革熱病毒(DV)血清型1、2、3及4造成之發炎性細胞激素釋出Example 15: Antagonistic anti-DVLR1/CLEC5A monoclonal antibody (mAbS) inhibits inflammatory cytokine release due to dengue virus (DV) serotypes 1, 2, 3 and 4.

用標準技術產生對抗DVLR1/CLEC5A之單株抗體。簡言之，將小鼠用DVLR-1.Fc融合蛋白質免疫，且雜交瘤藉由將得自小鼠之脾臟細胞與P3/NSI/1-Ag4-1[NS-1]骨髓瘤細胞(ATCC TIB-18)融合而形成。在所產生之mAbs中，9B12株、3E12亞株(3E12A2株、3E12C1株、3E12G9株)及8H8F5株於用DEN1(766733A系)、DEN2(PL046系)、DEN3(H-87系)及DEN4(866146A系)感染後，會以劑量-依存方式壓制TNF-α從巨噬細胞之釋出。參見圖15，其顯示由感染DV之CD14+巨噬細胞分泌入培養上清液之TNF-α之ELISA測量值。依照標準命名法，各抗體以分泌其之雜交瘤之株編號稱之。所以本發明亦提供分泌上述單株抗體之雜交瘤。Monoclonal antibodies against DVLR1/CLEC5A were generated using standard techniques. Briefly, mice were immunized with the DVLR-1.Fc fusion protein, and hybridomas were obtained by spleen cells from mice with P3/NSI/1-Ag4-1 [NS-1] myeloma cells (ATCC). TIB-18) is formed by fusion. Among the mAbs produced, 9B12 strain, 3E12 strain (3E12A2 strain, 3E12C1 strain, 3E12G9 strain) and 8H8F5 strain were used for DEN1 (766733A strain), DEN2 (PL046 strain), DEN3 (H-87 strain), and DEN4 ( 866146A) After infection, the release of TNF-α from macrophages is suppressed in a dose-dependent manner. See Figure 15, which shows ELISA measurements of TNF-[alpha] secreted into culture supernatant by CD14+ macrophages infected with DV. According to the standard nomenclature, each antibody is referred to as the number of the hybridoma cell from which it is secreted. Therefore, the present invention also provides a hybridoma which secretes the above-mentioned monoclonal antibodies.

該等結果證明抗-DVLR1/CLEC5A抗體在人類中將可做為治療劑，防止促發炎性細胞激素從DV所感染之CD14+巨噬細胞釋出。特定而言，但非限制性地，本實施例之單株抗體、或其片段、或與本實施例之抗體結合相同抗原決定部位之抗體(或其片段)可被調配成醫藥組合物，然後依照本文所提供之方法投與，以用於治療或預防人類DV感染。These results demonstrate that anti-DVLR1/CLEC5A antibodies will be used as therapeutic agents in humans to prevent the release of pro-inflammatory cytokines from DV-infected CD14+ macrophages. Specifically, but not by way of limitation, the monoclonal antibody of the present embodiment, or a fragment thereof, or an antibody (or a fragment thereof) that binds to the same epitope as the antibody of the present embodiment can be formulated into a pharmaceutical composition, and then It is administered in accordance with the methods provided herein for the treatment or prevention of human DV infection.

實施例16：測定免疫細胞上由登革熱病毒活化之模式識別受體(pattern recognition receptors，PRRs)Example 16: Determination of pattern recognition receptors (PRRs) activated by dengue virus on immune cells

樹狀細胞(DC)及巨噬細胞係DV感染之主要標靶(Halstead et al.,J.Exp.Med.1977,146：201-217；Palucka,Nat.Med.2000,6：748-749；Wu et al.,Nat.Med.2000,6：816-820)。雖然經感染之DCs會進行脫噬(任憑旁觀之DCs分泌促發炎性細胞激素)(Palmer et al.,J.Virol.2005,79,2432-2439)，經感染之巨噬細胞卻會存活至少45天並自感染後6小時起分泌多種細胞激素及化學激素(Chen et al.,J.Virol.2002,76：9877-9887)。此種情形暗示巨噬細胞為DV感染後之促發炎性細胞激素的主要來源，其中病毒粒子可能會藉由活化模式識別受體(PRRs)而啟動發炎反應。在此內容中，類鐸受體(TLRs)、C-型凝集素及類免疫球蛋白(類Ig)受體(如TREMs及類TREM受體(TIL))已經暗示為潛在之PRRs(Cook et al.,Nat.Immunol.2004,5,975-979；Klesney-Ttait et al.,Nat.Immunol.2006,7,1266-1273；Rovinson et al.,Nat.Immunol.2006,7,1258-1265)。Major targets for dendritic cells (DC) and macrophage cell line DV infection (Halstead et al., J. Exp. Med. 1977, 146: 201-217; Palucka, Nat. Med. 2000, 6: 748-749) Wu et al., Nat. Med. 2000, 6: 816-820). Although infected DCs undergo phagocytosis (withstand-by-side DCs secreting pro-inflammatory cytokines) (Palmer et al., J. Virol. 2005, 79, 2432-2439), infected macrophages survive at least. A variety of cytokines and chemical hormones were secreted for 45 days and 6 hours after infection (Chen et al., J. Virol. 2002, 76: 9987-9887). This situation suggests that macrophages are a major source of pro-inflammatory cytokines following DV infection, in which virions may initiate an inflammatory response by activating pattern recognition receptors (PRRs). In this context, terpenoid receptors (TLRs), C-type lectins, and immunoglobulin-like (Ig-like) receptors (such as TREMs and TREM-like receptors (TIL)) have been implicated as potential PRRs (Cook et Al., Nat. Immunol. 2004, 5, 975-979; Klesney-Ttait et al., Nat. Immunol. 2006, 7, 1266-1273; Rovinson et al., Nat. Immunol. 2006, 7, 1258-1265).

為判定登革熱病毒是否可結合並活化免疫細胞上之後選PRRs，使二十二種融合蛋白質在哺乳動物細胞中表現，並篩選其與DV2之交互作用(表2)。該等融合蛋白包含人類IgG1.Fc片段結合C-型凝集素及類Ig受體之胞外域。To determine whether dengue virus can bind and activate PRRs on immune cells, twenty-two fusion proteins were expressed in mammalian cells and screened for interaction with DV2 (Table 2). The fusion proteins comprise a human IgGl.Fc fragment that binds to the extracellular domain of a C-type lectin and an Ig-like receptor.

表2：重組受體.Fc融合蛋白質之構築 Table 2: Construction of recombinant receptor. Fc fusion protein

如圖16所示，DV與DLVR1/CLEC5A具有交互作用。特定言之，圖16a顯示由ELISA所測定之DV(5x10⁶ PFU)與DLVR1/CLEC5A(1 μg)之交互作用。在圖16b中，其以對抗DV套膜(E)蛋白質之mAb對DV(5x10⁶ PFU)與受體.Fc(5 μg)之複合物進行免疫沈澱並以西方轉漬偵測。圖16c顯示由ELISA所測定之EDTA(10 mM)對DLVR1/CLEC5A-DV交互作用的抑制。圖16d及16e顯示糖競爭檢定分析，其中DC-SIGN(CLEC4L)及DLVR1/CLEC5A兩者皆可增加DV對於人類293T細胞的結合(圖16d)，而糖之添加則以劑量-依存性之方式抑制生物素化DV對於經DC-SIGN-(左格)或DLVR1/CLEC5A-(右格)轉染之293T細胞的結合，如以流動式細胞計量術測定(圖16e)。根據圖16e，MFI表示平均螢光強度。單醣體(甘露糖及岩藻糖)及多糖體(甘露聚糖)之單位(U)分別為mM及mg/ml。圖16f顯示PNGaseF(500U)、DTT(0.1 M)、熱(95℃ 5分鐘)或UV(10 J/cm² )對於DLVR1/CLEC5A-DV交互作用的效應，如以ELISA所測定。數據係以三次獨立試驗之平均值±s.d.表示。進行雙尾學生t試驗。As shown in Figure 16, DV interacts with DLVR1/CLEC5A. Certain words, FIG. 16a shows DV (5x10 ⁶ PFU) as determined by the ELISA with the DLVR1 / CLEC5A (1 μg) of interaction. In Figure 16b, which is set against the film DV (E) mAb to the protein of DV (5x10 ⁶ PFU) receptor .Fc (5 μg) of the complexes were immunoprecipitated and Western blotted to detect. Figure 16c shows inhibition of DLVR1/CLEC5A-DV interaction by EDTA (10 mM) as determined by ELISA. Figures 16d and 16e show a sugar competition assay in which both DC-SIGN (CLEC4L) and DLVR1/CLEC5A increase DV binding to human 293T cells (Fig. 16d), while sugar addition is dose-dependent. Binding of biotinylated DV to DC-SIGN- (left panel) or DLVR1/CLEC5A- (right panel) transfected 293T cells was determined as determined by flow cytometry (Fig. 16e). According to Figure 16e, MFI represents the average fluorescence intensity. The units (U) of the monosaccharide (mannose and fucose) and the polysaccharide (mannan) are mM and mg/ml, respectively. Figure 16f shows the effect of PNGaseF (500 U), DTT (0.1 M), heat (95 °C 5 min) or UV (10 J/cm ² ) on DLVR1/CLEC5A-DV interaction as determined by ELISA. Data are expressed as the mean ± sd of three independent experiments. A two-tailed student t test was performed.

在所試驗之受體中，DC-SIGN已於先前顯示可與位於DV套膜(E)蛋白質上之聚醣產生交互作用(Pokidysheva,E.et al.,Cell 124：485-93(2006))。使用ELISA，DLVR1/CLEC5A.Fc(在DC-SIGN.Fc及DC-SIGNR.Fc之外)顯示可捕捉DV2(圖16a)。為確認DLVR1/CLEC5A與DV間交互作用之特異性，以蛋白質A瓊脂糖(Sepharose)珠粒免疫沈澱複合物，接著以抗-DV套膜(抗-E)單株抗體(mAb)進行探測。在DC-SIGN.Fc及DLVR1/CLEC5A.Fc之免疫沈澱物中偵測到E蛋白，因此確認DLVR1/CLEC5A可與登革熱病毒粒子產生交互作用(圖16b)。然而，儘管DC-SIGN對DV之結合為Ca⁺⁺ -依存性，EDTA(一種Ca⁺⁺ 螯合劑)對於DLVR1/CLEC5A-DV交互作用則並無效應(圖1c)。再者，以DC-SIGN及DLVR1/CLEC5A轉染293T細胞造成生物素化DV對該等細胞的結合增加(圖1d)。Among the receptors tested, DC-SIGN has previously been shown to interact with glycans located on the DV envelope (E) protein (Pokidysheva, E. et al., Cell 124:485-93 (2006) ). Using ELISA, DLVR1/CLEC5A.Fc (outside DC-SIGN.Fc and DC-SIGNR.Fc) was shown to capture DV2 (Fig. 16a). To confirm the specificity of the interaction between DLVR1/CLEC5A and DV, the complex was immunoprecipitated with Protein A Sepharose beads, followed by detection with an anti-DV envelope (anti-E) monoclonal antibody (mAb). The E protein was detected in the immunoprecipitates of DC-SIGN.Fc and DLVR1/CLEC5A.Fc, thus confirming that DLVR1/CLEC5A can interact with dengue virus particles (Fig. 16b). However, although the binding of DC-SIGN to DV is Ca ⁺⁺ -dependent, EDTA (a Ca ⁺⁺ chelating agent) has no effect on DLVR1/CLEC5A-DV interaction (Fig. 1c). Furthermore, transfection of 293T cells with DC-SIGN and DLVR1/CLEC5A resulted in increased binding of biotinylated DV to these cells (Fig. 1d).

在E蛋白質之Asn-67及Asn-153共有兩個保守性之N-連結性糖基化位點(Pokidysheva,E.et al.,Cell 124：485-93(2006))，且該等連附之聚醣已被證實涉及細胞連附及病毒進入(Modis,Y.et al.,J.Virol.79：1223-2131(2005))。為研究聚醣在DLVR1/CLEC5A與DV連結中之參與情形，使病毒粒子與岩藻糖、甘露糖、或甘露聚糖共同培育；其中至少兩種糖為DC-SIGN之配體(Mitchell te al.,J.Biol.Chem.276：28939-28945(2006))。如所預期，甘露糖及甘露聚糖造成DC-SIGN-DV交互作用之劑量-依存性抑制(圖16e)，而DLVR1/CLEC5A對DV之結合則在岩藻糖存在下顯著降低(p<00001)，且在甘露聚糖存在下有較低程度之降低(p=0.0005)。以PNGaseF對DV進行預處理亦顯著降低DLVR1/CLEC5A-DV交互作用(圖16f)，其建議存在於病毒E蛋白上之聚醣為結合所必要者。亦發現熱處理或二硫蘇糖醇(DTT)可遏止DLVR1/CLEC5A-DV交互作用(圖16f)，此建議聚醣在登革熱病毒粒子上之正確拓樸分布係重要的。There are two conserved N-linked glycosylation sites in Asn-67 and Asn-153 of E protein (Pokidysheva, E. et al., Cell 124:485-93 (2006)), and such linkages Attached glycans have been shown to be involved in cell attachment and viral entry (Modis, Y. et al., J. Virol. 79: 1223-2131 (2005)). To investigate the involvement of glycans in DLVR1/CLEC5A and DV linkages, virions were co-cultured with fucose, mannose, or mannan; at least two of the sugars were DC-SIGN ligands (Mitchell te al) J. Biol. Chem. 276: 28939-28945 (2006)). As expected, mannose and mannan caused dose-dependency inhibition of DC-SIGN-DV interaction (Fig. 16e), while DLVR1/CLEC5A binding to DV was significantly reduced in the presence of fucose (p<00001) ) and a lower degree of reduction in the presence of mannan (p=0.0005). Pretreatment of DV with PNGaseF also significantly reduced DLVR1/CLEC5A-DV interaction (Fig. 16f), suggesting that glycans present on the viral E protein are necessary for binding. Heat treatment or dithiothreitol (DTT) was also found to inhibit DLVR1/CLEC5A-DV interaction (Fig. 16f), suggesting that the correct topography of glycans on dengue virions is important.

實施例17：DC-SIGN及DVLR1/CLEC5A與登革熱病毒之交互作用Example 17: Interaction of DC-SIGN and DVLR1/CLEC5A with dengue virus

圖17顯示DC-SIGN及DVLR1/CLEC5A在人類PBMCs中之表現模式。以綴合有PE之抗-CD標記抗體(BD PharMingen)以及綴合有FITC之抗-DC-SIGN mAb(根據圖17a所示之具體例)或綴合有FITC之抗-DVLR1/CLEC5A mAb(R&D Systems)(根據圖17b所示之具體例)，對剛單離之PBMCs進行雙重染色。選通(gate)CD標記陽性細胞以測定DC-SIGN及DVLR1/CLEC5A(虛線)之表現。陰影區域代表同種型對照組。Figure 17 shows the expression patterns of DC-SIGN and DVLR1/CLEC5A in human PBMCs. An anti-CD-labeled antibody conjugated with PE (BD PharMingen) and an anti-DC-SIGN mAb conjugated with FITC (specific examples according to Figure 17a) or anti-DVLR1/CLEC5A mAb conjugated with FITC ( R&D Systems) (according to the specific example shown in Fig. 17b), double staining of newly isolated PBMCs. The CD labeled positive cells were gated to determine the performance of DC-SIGN and DVLR1/CLEC5A (dashed line). The shaded area represents the isotype control group.

DC-SIGN(其係表現於DCs及巨噬細胞上(圖17a))在其細胞質尾部含有三種基序，該等基序咸信涉及細胞吞噬作用或胞內運輸(Lozach et al.,J.Biol.Chem.2005,280,23698 23708)。相對的，DVLR1/CLEC5A則原始經辨識為僅表現在單核細胞及巨噬細胞上之DAP12-相關分子(圖17b)，然其配體及生物功能仍待測定(Bakker et al.,Proc.Natl.Acad.Sci.USA 1999,96,9792 9796)。DC-SIGN, which is expressed on DCs and macrophages (Fig. 17a), contains three motifs at its cytoplasmic tail that are involved in phagocytosis or intracellular transport (Lozach et al., J. Biol. Chem. 2005, 280, 23698 23708). In contrast, DVLR1/CLEC5A was originally identified as a DAP12-related molecule expressed only on monocytes and macrophages (Fig. 17b), but its ligand and biological functions remain to be determined (Bakker et al., Proc. USA 1999, 96, 9792 9796).

實施例18:DVLR1/CLEC5A對於DV誘導之DAP12磷醯化具必要性，但對於登革熱病毒之複製則不具必要性Example 18: DVLR1/CLEC5A is necessary for DV-induced DAP12 phosphorylation, but not necessary for replication of dengue virus

根據圖18所示之具體例，DVLR1/CLEC5A對於DV誘導之DAP12磷醯化具必要性，但對於登革熱病毒之複製則不具必要性。特定言之，在圖18中，其在西方轉漬分析上使用針對磷醯酪胺酸及DAP12之抗體，測定人類巨噬細胞中之DV誘導之DAP12磷醯化(2 h p.i.)。圖18b說明由DV及經UV-失活之DV(UV-DV)誘導之DAP12磷醯化的動力學。圖18c說明shRNAs使DVLR1/CLEC5A及DC-SIGN之表現下降(knock down)以及抑制DV-媒介(m.o.i.=5)之DAP12磷醯化的能力。圖18d顯示shRNAs對於巨噬細胞中之DV進入及複製的效應，如以流動式細胞計量術所測定。圖18e說明以共軛焦顯微鏡所檢驗到之抗-DVLR1/CLEC5A mAb、抗-DC-SIGN mAb、及小鼠IgG(50 μg/ml)對於非結構蛋白質NS3(紅色；經Cy3標記)表現的效應(Tassaneetrithep,B.etal.,J Exp Med 197：823-29(2003))。以Hoechst 33342(藍色)對比染色細胞。在圖18d及圖18e兩者中，巨噬細胞經DV感染(m.o.i.=5)以測定感染後48小時之NS3表現。圖18f顯示shRNAs對於經感染巨噬細胞之DV滴度的效應。According to the specific example shown in Fig. 18, DVLR1/CLEC5A is necessary for DV-induced DAP12 phosphorylation, but it is not necessary for the replication of dengue virus. Specifically, in Figure 18, DV12-induced DAP12 phosphorylation (2 h p.i.) in human macrophages was determined using Western blot analysis using antibodies against phosphonium tyrosine and DAP12. Figure 18b illustrates the kinetics of DAP12 phosphonium induced by DV and UV-inactivated DV (UV-DV). Figure 18c illustrates the ability of shRNAs to knock down the expression of DVLR1/CLEC5A and DC-SIGN and to inhibit DAP12 phosphorylation of DV-medium (m.o.i.=5). Figure 18d shows the effect of shRNAs on DV entry and replication in macrophages as determined by flow cytometry. Figure 18e illustrates the anti-DVLR1/CLEC5A mAb, anti-DC-SIGN mAb, and mouse IgG (50 μg/ml) as demonstrated by conjugated focus microscopy for non-structural protein NS3 (red; Cy3-labeled) Effect (Tassaneetrithep, B. et al., J Exp Med 197:823-29 (2003)). Cells were stained with Hoechst 33342 (blue) contrast. In both Fig. 18d and Fig. 18e, macrophages were infected with DV (m.o.i.=5) to determine NS3 performance 48 hours after infection. Figure 18f shows the effect of shRNAs on DV titers of infected macrophages.

發現以DV感染巨噬細胞可以劑量-依存性之方式誘發DAP12磷醯化(圖18a)。在活DV存在下，DAP12磷醯化在感染後(p.i.)12小時達到峰值並持續至少48小時，而經UV-失活之登革熱病毒(UV-DV)則只可啟動僅持續12小時之有限DAP12磷醯化(圖18b)，此指出在感染最初之2-6小時，DAP12磷醯化係與DV複製無關。DVLR1/CLEC5A之下降(knock down)(使用shRNA pLL3.7/DVLR1/CLEC5A會造成DAP12磷醯化之顯著減少，但DC-SIGN之下降(由pLL3.7/DC-SIGN造成)則否(圖18c)，此建議由DV引發之DAP12磷醯化係經由DVLR1/CLEC5A媒介。It was found that infection of macrophages with DV induced DAP12 phosphorylation in a dose-dependent manner (Fig. 18a). In the presence of live DV, DAP12 phosphorylation peaks at 12 hours post infection (pi) for at least 48 hours, whereas UV-inactivated dengue virus (UV-DV) can only be activated for only 12 hours. DAP12 phosphonium (Fig. 18b), indicating that DAP12 phosphonium is not associated with DV replication during the first 2-6 hours of infection. DVLR1/CLEC5A is knocked down (using shRNA pLL3.7/DVLR1/CLEC5A causes a significant decrease in DAP12 phosphorylation, but a decrease in DC-SIGN (caused by pLL3.7/DC-SIGN) no (Figure 18c), this suggests that the DAP12 phosphonium initiated by DV is via the DVLR1/CLEC5A mediator.

已知DC-SIGN涉及DV對於DCs之感染(Navarro-Sanchez et al.,EMBO Rep.4：723-28(2003)；Tassanetrothep et al.,J.Exp.Med.197：823-829(2003))。因此，藉由監測DV非結構蛋白質3(NS3)之表現(其係在DV於巨噬細胞中複製時表現)，試驗DVLR1/CLEC5A是否涉及DV進入巨噬細胞。與DC-SIGN相反，以shRNA使DVLR1/CLEC5A下降(圖18d)或是以抗-DVLR1/CLEC5A Ab阻斷DVLR1/CLEC5A-DV交互作用(圖18e)並不會抑制巨噬細胞中之NS3表現，如以流動式細胞計量術及共軛焦顯微鏡檢所分別檢驗。shRNA pLL3.7/DVLR1/CLEC5A亦無法抑制登革熱病毒粒子釋出至經感染巨噬細胞之上清液中，如以成斑檢定分析而測定(圖18f)。此等結果指出，DC-SIGN會中介DV感染及複製，而DV與DVLR1/CLEC5A之交互作用則會啟動細胞信號傳導。DC-SIGN is known to be involved in the infection of DCs by DV (Navarro-Sanchez et al., EMBO Rep. 4: 723-28 (2003); Tassanetrothep et al., J. Exp. Med. 197: 823-829 (2003) ). Thus, by monitoring the expression of DV non-structural protein 3 (NS3), which is expressed when DV replicates in macrophages, it was tested whether DVLR1/CLEC5A is involved in DV entry into macrophages. In contrast to DC-SIGN, DVLR1/CLEC5A decreased with shRNA (Fig. 18d) or DVLR1/CLEC5A-DV interaction with anti-DVLR1/CLEC5A Ab (Fig. 18e) did not inhibit NS3 expression in macrophages , for example, by flow cytometry and conjugated focus microscopy. shRNA pLL3.7/DVLR1/CLEC5A also failed to inhibit the release of dengue virus particles into the supernatant of infected macrophages as determined by plaque assay (Fig. 18f). These results indicate that DC-SIGN mediates DV infection and replication, and the interaction of DV with DVLR1/CLEC5A initiates cellular signaling.

實施例19：DVLR1/CLEC5A-登革熱病毒交互作用之抑制可壓抑經感染巨噬細胞之發炎反應而不影響病毒清除反應Example 19: Inhibition of DVLR1/CLEC5A-dengue virus interaction suppresses inflammatory response by infected macrophages without affecting viral clearance response

為測定DVLR1/CLEC5A是否涉及DV誘導之發炎反應，檢驗巨噬細胞在DV感染後之發炎性細胞激素分泌。To determine whether DVLR1/CLEC5A is involved in a DV-induced inflammatory response, macrophage secretion of inflammatory cytokines following DV infection was examined.

根據圖19所示之具體例，DVLR1/CLEC5A對於DV-媒介之TNF-α分泌相當必要，但對INF-α分泌則否。更特定言之，圖19a說明巨噬細胞之DV及UV-DV誘導性TNF-α分泌的劑量-依存性，如以ELISA在6 h及12 h p.i.測量。圖19b說明DV感染(m.o.i.=5)後之INF-α表現的動力學。圖19c說明DVLR1/CLEC5A及DC-SIGN shRNA對於TNF-α、IL-6、MIP1-α、IL-8、IP-10及INF-α自經DV感染(m.o.i.=5)之巨噬細胞分泌的效應。在圖19d中，以受體特異性shRNA所進行之下降(knock down)實驗說明，DV誘導之INF-α分泌係經由TLR7-MyD88途徑而TNF-α分泌係經由DVLR1/CLEC5A-TLR7-MyD88途徑。圖19e說明可抑制反應DV血清型1-4之TNF-α分泌的桔抗性抗-DVLR1/CLEC5AmAbs受到抑制(參見表3)。According to the specific example shown in Fig. 19, DVLR1/CLEC5A is essential for DV-mediated TNF-α secretion, but not for INF-α secretion. More specifically, Figure 19a illustrates the dose-dependency of DV and UV-DV-induced TNF-α secretion by macrophages as measured by ELISA at 6 h and 12 h p.i. Figure 19b illustrates the kinetics of INF-alpha expression after DV infection (m.o.i.=5). Figure 19c illustrates the secretion of DVLR1/CLEC5A and DC-SIGN shRNA from TNF-α, IL-6, MIP1-α, IL-8, IP-10 and INF-α from DV-infected (moi=5) macrophages. effect. In Figure 19d, the knockdown experiments with receptor-specific shRNAs indicate that DV-induced INF-alpha secretion via the TLR7-MyD88 pathway and TNF-alpha secretion via the DVLR1/CLEC5A-TLR7-MyD88 pathway . Figure 19e illustrates inhibition of citrus-resistant anti-DVLR1/CLEC5 AmAbs that inhibit TNF-alpha secretion by reaction DV serotypes 1-4 (see Table 3).

表3：抗-人類DVLR1/CLEC5A mAbs之特徵。"*"-p<0.05；"**"-p<0.01；"***"-p<0.001。抗體(10 μg/樣本)可抑制由人類巨噬細胞之DV誘導性TNF-α分泌。進行雙尾學生t試驗並將數據與各個適當之同種型控制組抗體比較。可啟動由人類巨噬細胞之TNF-α分泌(相較於同種型控制組)的抗體定義為催動性。特異性由使用pLL3.7/DVLR1/CLEC5A-shRNA遏止TNF-α分泌而確認(pLL3.7載體及pLL3.7/DC-SIGN對於抗體-媒介之TNF-α分泌並無效應)。ELISA，酶鍵連免疫吸附檢定分析；WB，西方轉漬；FACS，螢光活化細胞分選。抗體係不含疊氮化物、經無菌過濾、且具有低於每毫克0.1EU之內毒素量。 Table 3: Characteristics of anti-human DVLR1/CLEC5A mAbs. "*"-p<0.05;"**"-p<0.01;"***"-p<0.001. Antibodies (10 μg/sample) inhibited DV-induced TNF-α secretion by human macrophages. A two-tailed Student's t-test was performed and the data was compared to each appropriate isotype control group antibody. Antibodies that initiate TNF-[alpha] secretion by human macrophages (as compared to the isotype control group) are defined as priming. Specificity was confirmed by inhibition of TNF-α secretion by using pLL3.7/DVLR1/CLEC5A-shRNA (pLL3.7 vector and pLL3.7/DC-SIGN had no effect on antibody-mediated TNF-α secretion). ELISA, enzyme-linked immunosorbent assay; WB, Western blotting; FACS, fluorescence activated cell sorting. The anti-system is free of azide, sterile filtered, and has an endotoxin level of less than 0.1 EU per mg.

使用M.R.mAb(抗-甘露糖受體mAb；mIgG1)及小鼠IgM(mIgM)作為陰性控制組。在圖19d及圖19e兩者中，以DV感染巨噬細胞(m.o.i.=5)，並在36 h p.i.收集進行細胞激素檢定分析。數據係以三次獨立試驗(使用來自至少三位不同捐血者之材料進行)之平均值±s.d.表示。進行雙尾學生t試驗。"ND"表示未偵測。M.R. mAb (anti-mannose receptor mAb; mIgG1) and mouse IgM (mIgM) were used as negative control groups. In both Fig. 19d and Fig. 19e, macrophages (m.o.i.=5) were infected with DV, and cytokine assays were performed at 36 h p.i. Data are expressed as the mean ± s.d. of three independent experiments (using materials from at least three different donors). A two-tailed student t test was performed. "ND" means not detected.

在6 h p.i.，偵測到劑量-依存性之TNF-α分泌，其中經DV或UV-DV感染之巨噬細胞分泌類似量之細胞激素(圖19a，左格)。然而，在12 h p.i.，DV會進一步增加TNF-α分泌，而UV-DV則不會(圖19a，右格)。在48 h之時程中，經DV感染之巨噬細胞的TNF-α分泌持續增加，而在以UV-DV感染後24 h-48 h，此種細胞激素幾乎無法偵測到(圖19b)。此等數據符合DAP12之動力學(圖18b)，因而建議DV媒介之TNF-α分泌係與DAP12之活化相關。其亦觀察到，DVLR1/CLEC5A下降(knock down)會以大於DC-SIGN下降之程度，抑制TNF-α、IL-6、IL-8、MIP1-α、及IP-10自經DV感染之巨噬細胞釋出(圖19c)。然而，pLL3.7/DC-SIGN可輕微抑制INF-α之分泌(p=0.048)，而pLL3.7/DVLR1/CLEC5A則對INF-α無作用(圖19c)。At 6 h p.i., dose-dependent TNF-α secretion was detected, in which macrophages infected with DV or UV-DV secrete similar amounts of cytokines (Fig. 19a, left panel). However, at 12 h p.i., DV further increased TNF-α secretion, whereas UV-DV did not (Fig. 19a, right panel). During the 48 h time course, the secretion of TNF-α by DV-infected macrophages continued to increase, and this cytokine was almost undetectable 24 h to 48 h after UV-DV infection (Fig. 19b). . These data are consistent with the kinetics of DAP12 (Fig. 18b) and it is therefore suggested that the TNF-[alpha] secretion line of the DV vector is associated with activation of DAP12. It has also been observed that DVLR1/CLEC5A knockdown will inhibit TNF-α, IL-6, IL-8, MIP1-α, and IP-10 from DV infection by a greater than DC-SIGN decline. Phagocyte release (Fig. 19c). However, pLL3.7/DC-SIGN slightly inhibited the secretion of INF-α (p=0.048), while pLL3.7/DVLR1/CLEC5A had no effect on INF-α (Fig. 19c).

為進一步了解導致細胞激素分泌之由DV活化之信號傳導途徑，在DV感染之前，以shRNAs轉染巨噬細胞，以使DVLR1/CLEC5A、DC-SIGN、TLR4、TLR7或MyD88下降。所得之數據指出，DV誘導之INF-α分泌係經由TLR7-MyD88途徑產生(p=0.0016)，而TNF-α分泌則係由DVLR1/CLEC5A(p=0.0013)及TLR7-MyD88(p=0.013)兩者媒介(圖19d)。產生一組抗-DVLR1/CLEC5A mAbs，其對於DV之四種血清型具有不同之拮抗效應(見上文表3)，如以自經DV感染之巨噬細胞之TNF-α分泌的抑制所測定(圖3e)。此等數據指出，儘管DVLR1/CLEC5A之不同抗原決定部位似乎媒介個別之交互作用，能夠抑制DVLR1/CLEC5A之抗體卻可壓抑經相關DV血清型感染之巨噬細胞的發炎反應。抗-DVLR1/CLEC5A mAbs之不同桔抗效應可能係與各DV血清型係和DVLR1/CLEC5A之迥異抗原決定部位結合的事實有關，而抗-DVLR1/CLEC5A mAb可抑制結合位點與抗-DVLR1/CLEC5A mAb之結合位點重疊之特異DV血清型的結合。To further understand the signaling pathways responsible for DV activation leading to cytokine secretion, macrophages were transfected with shRNAs prior to DV infection to reduce DVLR1/CLEC5A, DC-SIGN, TLR4, TLR7 or MyD88. The data obtained indicated that DV-induced INF-α secretion was produced via the TLR7-MyD88 pathway (p=0.0016), whereas TNF-α secretion was from DVLR1/CLEC5A (p=0.0013) and TLR7-MyD88 (p=0.013). Both media (Figure 19d). A set of anti-DVLR1/CLEC5A mAbs were generated which have different antagonistic effects on the four serotypes of DV (see Table 3 above), as determined by inhibition of TNF-[alpha] secretion from DV-infected macrophages (Figure 3e). These data indicate that although the different epitopes of DVLR1/CLEC5A appear to mediate individual interactions, antibodies that inhibit DVLR1/CLEC5A can suppress the inflammatory response of macrophages infected with the relevant DV serotype. The different orange anti-effects of anti-DVLR1/CLEC5A mAbs may be related to the fact that each DV serotype and the distinct epitope of DVLR1/CLEC5A bind, while anti-DVLR1/CLEC5A mAb inhibits the binding site and anti-DVLR1/ Binding of specific DV serotypes where the binding sites of the CLEC5A mAb overlap.

實施例20：由抗體依存性增強(antibody-dependentExample 20: Enhanced antibody-dependent (antibody-dependent) enhancement,ADE)媒介之INF-α分泌與DVLR1/CLEC5A無依存性Enhancement, ADE) media INF-α secretion and DVLR1/CLEC5A non-dependency

先前已經證明，非中和性抗-DV Abs可促進DV經由FcR受體進入標靶細胞，並因而增進細胞激素釋放(Halstead et al.,J.Exp.Med.146：201-217(1977)；Goncalvez et al.,Proc Natl Acad Sci USA 104：=9422-9427(2007))，此係稱為感染之抗體依存性增強(ADE)的現象。舉例而言，抗-prM及抗-E mAb已經顯示可在試管內誘導此效應(Huang et al.,J Immuno 176：2825-2832(2006))。在此，針對DVLR1/CLEC5A-DV交互作用之封阻是否可抑制ADE而進行研究。It has previously been demonstrated that non-neutralizing anti-DV Abs can promote DV entry into target cells via FcR receptors and thereby increase cytokine release (Halstead et al., J. Exp. Med. 146:201-217 (1977) Goncalvez et al., Proc Natl Acad Sci USA 104:=9422-9427 (2007)), which is referred to as the phenomenon of antibody-dependent enhancement (ADE) of infection. For example, anti-prM and anti-E mAb have been shown to induce this effect in vitro (Huang et al., J Immuno 176:2825-2832 (2006)). Here, whether or not the blocking of the DVLR1/CLEC5A-DV interaction can suppress ADE is investigated.

根據圖20所示之具體例，DVLR1/CLEC5A對於ADE媒介之TNF-α分泌具必要性，但對於INF-α分泌則不具必要性。更特定言之，在圖20a中，其以DV(m.o.i.=5)、DV/抗-E、或DV/抗-prM免疫複合物(ADE)感染巨噬細胞36h，接著以抗-NS3 mAb偵測DV複製。在拮抗性抗-DVLR1/CLEC5A mAb(1 μg；純系9B12H4)或同種型控制組存在下，以DV2(圖20b)、或是DV/抗-prM或DV/抗-E複合物(圖20c)感染取自10名個體之巨噬細胞。以ELISA測定TNF-α及INF-α分泌。進行雙尾學生t試驗。According to the specific example shown in Fig. 20, DVLR1/CLEC5A is necessary for TNF-α secretion by ADE media, but it is not necessary for INF-α secretion. More specifically, in Figure 20a, it infects macrophages with DV (moi=5), DV/anti-E, or DV/anti-prM immune complex (ADE) for 36 h, followed by anti-NS3 mAb Measure DV replication. In the presence of antagonistic anti-DVLR1/CLEC5A mAb (1 μg; pure line 9B12H4) or isotype control group, DV2 (Fig. 20b), or DV/anti-prM or DV/anti-E complex (Fig. 20c) Infected macrophages from 10 individuals. TNF-α and INF-α secretion were measured by ELISA. A two-tailed student t test was performed.

在抗-DVLR1/CLEC5A mAb(或同種型控制組)存在下，單以DV或是結合抗-prM/DV或抗-E/DV免疫複合物而感染原代人類巨噬細胞36 h。發現相較於僅使用DV者，抗-PrM/DV或抗-E/DV免疫複合物(ADE)可增加NS3之表現(圖20a)以及TNF-α及INF-α之分泌量(圖20b及20c)。然而，在抗-DVLR1/CLEC5A mAb顯著抑制由經DV、抗-prM/DV、及抗-E/DV免疫複合物感染之巨噬細胞的TNF-α分泌時(圖20c)，INF-α之分泌卻不受影響，因此建議由ADE媒介之INF-α分泌與DVLR1/CLEC5A無依存性(與上文關於DV誘導之INF-α生產所註記者相同)。Primary human macrophages were infected with DV alone or in combination with anti-prM/DV or anti-E/DV immune complexes for 36 h in the presence of anti-DVLR1/CLEC5A mAb (or isotype control group). It was found that anti-PrM/DV or anti-E/DV immune complex (ADE) increased the performance of NS3 (Fig. 20a) and the secretion of TNF-α and INF-α compared with those using only DV (Fig. 20b and 20c). However, when anti-DVLR1/CLEC5A mAb significantly inhibited TNF-α secretion by macrophages infected with DV, anti-prM/DV, and anti-E/DV immune complexes (Fig. 20c), INF-α Secretion is unaffected, so it is recommended that INF-alpha secretion by ADE media is not dependent on DVLR1/CLEC5A (same as the reporter above for DV-induced INF-alpha production).

實施例21:DVLR1/CLEC5A在登革熱病毒誘導之血管滲漏中的參與Example 21: Participation of DVLR1/CLEC5A in dengue virus-induced vascular leakage

DHF及DSS之特點係血漿滲漏以及皮下及生命器官(vital organ)出血。此等症狀係由免疫細胞釋出諸多可溶性媒介分子及細胞激素而增加血管通透性所造成(Green et al.,Curr.Opin.Infect.Dis.19：429-436(2006))。為測定DVLR1/CLEC5A是否涉及DV誘導之血管滲漏，將人類真皮微血管內皮細胞(HMEC-1)之單層用於通透性檢定分析中(Carr et al.,J.Med.Virol.69：521-528(2003))。DHF and DSS are characterized by plasma leakage and bleeding from the subcutaneous and vital organs. These symptoms are caused by the release of many soluble mediators and cytokines by immune cells to increase vascular permeability (Green et al., Curr. Opin. Infect. Dis. 19: 429-436 (2006)). To determine whether DVLR1/CLEC5A is involved in DV-induced vascular leakage, a single layer of human dermal microvascular endothelial cells (HMEC-1) was used for permeability assays (Carr et al., J. Med. Virol. 69: 521-528 (2003)).

根據圖21所示之具體例，桔抗性抗-DVLR1/CLEC5A mAbs可拯救經DV感染之巨噬細胞上清液對內皮細胞單層所造成之通透化。更特定言之，圖21a說明在與取自經DV或DV/抗-PrM複合物(ADE)感染之巨噬細胞的上清液進行培育後，由HRP遷移測量所測定之HMEC-1單層之通透性隨時間之變化。上清液中之TNF-α含量以ELISA測量。如圖21b所示，測定TNFR2.Fc(5 μg/ml)及抗-DVLR1/CLEC5A(純系9B12H4；5 μg/ml)對於內皮細胞單層通透化之抑制作用。數據係以三次獨立試驗之平均值±s.d.表示。進行雙尾學生t試驗。According to the specific example shown in Fig. 21, the orange-resistant anti-DVLR1/CLEC5A mAbs can rescue the permeabilization of the endothelial cell monolayer by the DV-infected macrophage supernatant. More specifically, Figure 21a illustrates the HMEC-1 monolayer as determined by HRP migration measurements after incubation with supernatants from macrophages infected with DV or DV/anti-PrM complex (ADE). The permeability changes with time. The TNF-α content in the supernatant was measured by ELISA. As shown in Fig. 21b, the inhibitory effect of TNFR2.Fc (5 μg/ml) and anti-DVLR1/CLEC5A (pure line 9B12H4; 5 μg/ml) on endothelial cell monolayer permeabilization was determined. Data are expressed as the mean ± s.d. of three independent experiments. A two-tailed student t test was performed.

發現取自經DV或抗-prM/DV免疫複合物感染之巨噬細胞的上清液可誘導HMEC-1單層之通透性；其中在感染之最初36 h-48 h，免疫復合物(ADE)可產生較單獨之DV更為顯著之效應(圖21a，左)。再者，盡管重組TNFR2.Fc之TNF-α中和亦可部分抑制由DV或抗-prM/DV所啟動之通透性誘導(p<0.005)(圖21b)，抗-DVLR1/CLEC5A mAb在此方面卻更為有效(圖21b)。其觀察到，在TNF-α之外，抗-DVLR1/CLEC5A尚可封阻巨噬細胞之其他發炎性細胞激素分泌，此可能可解釋此種現象。Supernatants from macrophages infected with DV or anti-prM/DV immune complexes were found to induce permeability of the HMEC-1 monolayer; in the first 36 h to 48 h of infection, immune complexes ( ADE) produces a more pronounced effect than a separate DV (Fig. 21a, left). Furthermore, although TNF-α neutralization of recombinant TNFR2.Fc also partially inhibited permeability induction initiated by DV or anti-prM/DV (p < 0.005) (Fig. 21b), anti-DVLR1/CLEC5A mAb was This aspect is more effective (Figure 21b). It has been observed that in addition to TNF-[alpha], anti-DVLR1/CLEC5A can block the secretion of other inflammatory cytokines by macrophages, which may explain this phenomenon.

實施例22：拮抗性mAbs與登革熱病毒誘導之TNF-α分泌問的劑量-依存***互作用Example 22: Dose-dependency interaction between antagonist mAbs and dengue virus-induced TNF-α secretion

針對DV-CLEC5A交互作用的封阻是否可在活體內拯救小鼠不受DV誘導之死亡而進行進一步之研究。根據圖22所示之具體例，其顯示mDVLR1/CLEC5A與DV之交互作用(圖22a)以及mDVLR1/CLEC5A在小鼠細胞中之表現模式(圖22b及22c)。更特定言之，圖22a顯示由ELISA所測定之DV(5x10⁶ PFU)與人類及小鼠DLVR1/CLEC5A.Fc(1 μg)的交互作用。選通(gate)F4/80及CD標記陽性細胞以測定mDVLR1/CLEC5A在小鼠脾細胞(圖22b)、小鼠骨髓(BM)-衍生性巨噬細胞及小鼠似巨噬細胞Raw264.7細胞(圖22b及22c)中之表現。Further studies were conducted on whether blocking of DV-CLEC5A interaction can rescue mice from DV-induced death in vivo. According to the specific example shown in Fig. 22, it shows the interaction of mDVLR1/CLEC5A with DV (Fig. 22a) and the expression pattern of mDVLR1/CLEC5A in mouse cells (Fig. 22b and 22c). More specific words, FIG. 22a show DV (5x10 ⁶ PFU) as determined by the ELISA interacting with the mouse and human DLVR1 / CLEC5A.Fc (1 μg) of. Gate F4/80 and CD-labeled positive cells to determine mDVLR1/CLEC5A in mouse spleen cells (Fig. 22b), mouse bone marrow (BM)-derived macrophages, and mouse-like macrophages Raw264.7 Performance in cells (Figures 22b and 22c).

發現小鼠DVLR1/CLEC5A(mDVLR1/CLEC5A)可以類似人類DVLR1/CLEC5A之親和力結合DV(圖22a)，且mDVLR1/CLEC5A表現於骨髓細胞譜系(CD11b+，F4/80+)、骨髓衍生性巨噬細胞及小鼠似巨噬細胞Raw264.7細胞(圖22b及22c)。Mouse DVLR1/CLEC5A (mDVLR1/CLEC5A) was found to bind DV similarly to the affinity of human DVLR1/CLEC5A (Fig. 22a), and mDVLR1/CLEC5A was expressed in the bone marrow cell lineage (CD11b+, F4/80+), bone marrow-derived macrophages Mouse-like macrophage Raw264.7 cells (Fig. 22b and 22c).

實施例23：mDVLR1/CLEC5A-DV交互作用之封阻可壓抑自Raw264.7細胞之DV誘導性TNF-α分泌Example 23: Blockade of mDVLR1/CLEC5A-DV interaction can suppress DV-induced TNF-α secretion from Raw264.7 cells

根據圖23所示之具體例，mDVLR1/CLEC5A-DV交互作用之封阻可壓抑自Raw264.7細胞之DV誘導性TNF-α分泌。更特定言之，圖23a顯示人類DC-SIGN可增加DV與小鼠巨噬細胞細胞系Raw264.7之結合，並增強DV感染之刺激效應。以ELISA測定TNF-α之釋出。圖23b顯示拮抗性mAbs對於小鼠DVLR1/CLEC5A之辨識。在mAbs之存在下，使之存在下，使穩定表現人類DC-SIGN之Raw264.7細胞與DV2(PL046；m.o.i.=30)共同進行培育。以ELISA測定上清液中之TNF-α量(在48 h p.i.)。圖23c顯示抗-DVLR1/CLEC5A mAbs(純系：3D2H6及10D7H3)可以劑量-依存性之方式抑制DV2-(NGC-N；m.o.i.=30)誘導之TNF-α釋出。以mIgG1作為同種型相符之陰性控制組。According to the specific example shown in Figure 23, the blockade of mDVLR1/CLEC5A-DV interaction can suppress DV-induced TNF-α secretion from Raw264.7 cells. More specifically, Figure 23a shows that human DC-SIGN increases the binding of DV to the mouse macrophage cell line Raw264.7 and enhances the stimulatory effects of DV infection. The release of TNF-α was measured by ELISA. Figure 23b shows the recognition of antagonist mAbs for mouse DVLR1/CLEC5A. Raw264.7 cells stably expressing human DC-SIGN were co-cultured with DV2 (PL046; m.o.i.=30) in the presence of mAbs in the presence of mAbs. The amount of TNF-α in the supernatant was determined by ELISA (at 48 h p.i.). Figure 23c shows that anti-DVLR1/CLEC5A mAbs (pure lines: 3D2H6 and 10D7H3) inhibited DV2-(NGC-N; m.o.i.=30) induced TNF-α release in a dose-dependent manner. The mIgG1 was used as a negative control group of the same type.

DV會刺激穩定表現人類DC-SIGN之Raw264.7細胞(Raw264.7/DC-SIGN)分泌TNF-α(圖23a)，而以拮抗性mAbs(表4)封阻mDVLR1/CLEC5ADV交互作用可以劑量-依存性之方式遏止Raw264.7/DC-SIGN細胞之DV誘導性TNF-α分泌(圖23b及23c)。DV stimulates the stable expression of human DC-SIGN Raw264.7 cells (Raw264.7/DC-SIGN) to secrete TNF-α (Fig. 23a), while antagonistic mAbs (Table 4) block mDVLR1/CLEC5ADV interaction. - DV-inducible TNF-[alpha] secretion by Raw264.7/DC-SIGN cells was stopped in a Dependent manner (Figures 23b and 23c).

表4：抗-小鼠DVLR1/CLEC5A mAbs之特徵。"*"-p<0.05；"**"-p<0.01；"ND"-未進行。抗體可抑制由小鼠巨噬細胞細胞系Raw264.7/DC-SIGN之DV誘導性TNF-α分泌。進行雙尾學生t試驗並將數據與各個適當之同種型控制組抗體比較。ELISA，酶鍵連免疫吸附檢定分析；FACS，螢光活化細胞分選。抗體係不含疊氮化物、經無菌過濾、且具有低於每毫克0.1EU之內毒素量。 Table 4: Characteristics of anti-mouse DVLR1/CLEC5A mAbs. "*"-p<0.05;"**"-p<0.01;"ND"-not performed. The antibody inhibits DV-inducible TNF-α secretion by the mouse macrophage cell line Raw264.7/DC-SIGN. A two-tailed Student's t-test was performed and the data was compared to each appropriate isotype control group antibody. ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting. The anti-system is free of azide, sterile filtered, and has an endotoxin level of less than 0.1 EU per mg.

實施例24：登革熱病毒(NGC-N)可誘導STAT1-/-小鼠之死亡Example 24: Dengue virus (NGC-N) induces death in STAT1-/- mice

根據圖24所示之具體例，以一劑量範圍(自102至105 PFU)之DV2/PL046或DV2/NGC-N系(i.p.及i.c.途徑)挑戰STAT1-/-小鼠(n=5/組)4週。數據以Kaplan-Meier存活曲線表示。According to the specific example shown in Figure 24, STAT1-/- mice were challenged with a dose range (from 102 to 105 PFU) of DV2/PL046 or DV2/NGC-N lines (ip and ic pathways) (n=5/group) )4 weeks. Data are expressed as Kaplan-Meier survival curves.

INF-α可作用而抑制經感染及未經感染細胞兩者中之病毒複製，而對於DV感染之INF媒介性反應同時涉及STAT1-依存性途徑(對於病毒複製之控制為必要)及STAT1-無依存性途徑(對於感染之消散為必要)兩者(Shresta et al.,J.Immunol.175：3946-3954(2005))。儘管野生型之小鼠對於DV感染具有抗性，STAT1缺陷(STAT1 ^-/- )(Durbin et al.,Cell 84：443-450(1996))小鼠對於DV2-9(New Guinea C-N系)誘導之死亡則為敏感(圖24)。INF-α acts to inhibit viral replication in both infected and uninfected cells, whereas the INF vector response to DV infection involves both the STAT1-dependent pathway (necessary for viral replication control) and STAT1-Non Dependent pathways (necessary for the dissipation of infection) are both (Shresta et al., J. Immunol. 175: 3946-3954 (2005)). Although wild-type mice are resistant to DV infection, STAT1 deficiency ( STAT1 ^-/- ) (Durbin et al., Cell 84: 443-450 (1996)) mice are induced for DV2-9 (New Guinea CN). The death is sensitive (Figure 24).

實施例25：拮抗性mAbs對抗DVLR1/CLEC5A之潛在治疲效應Example 25: Potential anti-aging effects of antagonist mAbs against DVLR1/CLEC5A

針對桔抗性mAbs對STAT1 ^-/- 小鼠之潛在治療效應進行進一步試驗。根據圖25所示之具體例，抗-DVLR1/CLEC5A mAbs可在STAT1缺陷小鼠體內預防DV誘導之血管滲漏及死亡。更特定言之，在圖25a中，對抗小鼠DVLR1/CLEC5A培育之mAb 3D2H6可抑制經DV挑戰之STAT1 ^-/- 小鼠的皮下及腸道出血。在圖25b中，對抗DVLR1/CLEC5A之mAb(3D2H6及10D7H3)可減少經DV挑戰之STAT1 ^-/- 小鼠的血漿滲漏進入生命器官，如以Evans藍檢定分析所測定。圖25c說明藉著自器官萃取Evans藍而進行之血管通透性定量。數據係以三次獨立試驗之平均值±s.d.表示：*p<0.05；**p<0.01；***p<0.001(學生t試驗)。圖25d說明於有及無抗-DVLR1/CLEC5A mAbs或TNFR2.Fc存在下，經DV挑戰之STAT1 ^-/- 小鼠在p.i.第7天之TNF-α及IP-10之血清含量(n=8；上及中)以及病毒滴度(n=4；下)。進行雙尾學生t試驗。圖25e說明在拮抗性抗-DVLR1/CLEC5A mAbs或TNFR2.Fc存在下，經DV2(New Guinea C-N系，1x10⁵ PFU/小鼠，i.p.加i.c.途徑)挑戰之STAT1缺陷小鼠的存活曲線。數據係取自四次獨立試驗(每組各17隻小鼠)，並以Kaplan-Meier存活曲線並使用指數系列法(log rank test)表示。指出對於DVLR1/CLEC5A mAbs及小鼠IgG治療間之顯著差異的p 值。Further trials were conducted on the potential therapeutic effects of citrus-resistant mAbs on STAT1 ^-/- mice. According to the specific example shown in Figure 25, anti-DVLR1/CLEC5A mAbs prevent DV-induced vascular leakage and death in STAT1-deficient mice. More specifically, in Figure 25a, mAb 3D2H6 incubated against mouse DVLR1/CLEC5A inhibited subcutaneous and intestinal bleeding in DV challenged STAT1 ^-/- mice. In Figure 25b, mAbs against DVLR1/CLEC5A (3D2H6 and 10D7H3) reduced plasma leakage of DV-challenged STAT1 ^-/- mice into vital organs as determined by Evans Blue assay. Figure 25c illustrates vascular permeability quantification by extracting Evans blue from an organ. Data are expressed as the mean ± sd of three independent experiments: *p <0.05; **p <0.01; ***p < 0.001 (student t test). Figure 25d illustrates the serum levels of TNF-α and IP-10 on day 7 of pi in DV-challenged STAT1 ^-/- mice in the presence and absence of anti-DVLR1/CLEC5A mAbs or TNFR2.Fc (n=8) ; upper and middle) and virus titer (n=4; bottom). A two-tailed student t test was performed. FIG. 25e described in antagonistic anti -DVLR1 / CLEC5A mAbs or TNFR2.Fc present, by STAT1 (New Guinea CN-based, 1x10 ⁵ PFU / mouse, ip route add ic) challenge survival curves of mice DV2 defects. Data were taken from four independent experiments (17 mice per group) and expressed as Kaplan-Meier survival curves using a log rank test. P values were noted for significant differences between DVLR1/CLEC5A mAbs and mouse IgG treatment.

在p.i.第8天，除皮下及腸道出血之外，經DV挑戰之STAT1 ^-/- 小鼠尚有毛髮摺皺(Ruffled fur)及輕微之癱瘓(圖25a)，且在感染7-14天內皆死亡(圖25e)。在p.i.第0、1、3、5及7天，投與5劑之Abs(100 μg/小鼠，i.p.)或TNFR2.Fc(100 μg/小鼠，i.p.)。在p.i.第9天，經抗-DVLR1/CLEC5A mAbs治療小鼠體內滲漏進入經DV挑戰小鼠腎臟、肝臟、胃、小腸、大腸、及胰臟之Evans藍量相較於控制組顯著減少(圖25b及25c)。抗-DVLR1/CLEC5A mAbs亦顯著降低TNF-α及IP-10之血清含量(圖25d；上及中)，且在p.i.第7天未壓抑病毒複製(圖25d；下)，並在p.i.第14天保護小鼠不會死亡(70%保護率)。在p.i.第21天所觀察到之經抗-DVLR1/CLEC5A mAbs治療小鼠的整體存活率為48%(圖25e)，且在p.i.第23天DV自存活小鼠之血清中清除(數據未示)。因此，DVLR1/CLEC5A-DV交互作用之封阻似乎可防止與DV相關之出血及血漿滲漏併發症，並可壓抑巨噬細胞發炎反應，且不會損害適應性免疫系統之病毒清除。相對的，TNFR2.Fc既不可降低血管通透性(圖25c)，亦不可保護小鼠不會死亡(圖25e)，儘管其可有效降低TNF-α之血清含量(圖25d)。On the 8th day of pi, in addition to subcutaneous and intestinal bleeding, DV-challenged STAT1 ^-/- mice still had hair folds (Ruffled fur) and mild sputum (Fig. 25a), and within 7-14 days of infection Both died (Fig. 25e). Five doses of Abs (100 μg/mouse, ip) or TNFR2.Fc (100 μg/mouse, ip) were administered on days 0, 1, 3, 5 and 7 of pi. On day 9 of pi, the amount of Evans blue in the kidneys, liver, stomach, small intestine, large intestine, and pancreas of DV challenge mice was significantly reduced in mice treated with anti-DVLR1/CLEC5A mAbs compared with the control group ( Figures 25b and 25c). Anti-DVLR1/CLEC5A mAbs also significantly reduced serum levels of TNF-α and IP-10 (Fig. 25d; upper and middle), and did not suppress viral replication on day 7 of pi (Fig. 25d; bottom), and at pi 14th Days protect mice from death (70% protection). The overall survival rate of mice treated with anti-DVLR1/CLEC5A mAbs observed on day 21 of pi was 48% (Fig. 25e), and was cleared from the serum of surviving mice on day 23 of pi (data not shown) ). Therefore, the blockade of DVLR1/CLEC5A-DV interaction appears to prevent DV-related bleeding and plasma leakage complications, and can suppress macrophage inflammatory responses without compromising viral clearance by the adaptive immune system. In contrast, TNFR2.Fc neither reduced vascular permeability (Fig. 25c) nor protected mice from death (Fig. 25e), although it was effective in reducing serum levels of TNF-α (Fig. 25d).

實施例26：DVLR1/CLEC5A涉及JEV媒介之DAP12磷酸化作用及自人類巨噬細胞之TNF-α分泌Example 26: DVLR1/CLEC5A is involved in DAP12 phosphorylation of JEV mediators and TNF-alpha secretion from human macrophages

和DV相似，JEV具有類似之病毒感染反應模式，其咸信在所有黃病毒中皆為相同或相似。如圖26a所示，分別以ELISA測定DVLR1/CLEC5A(1 μg)與JEV及DV(5x10⁶ PFU)之交互作用。DV與人類DVLR1/CLEC5A(長度187胺基酸)序列編號：72具交互作用，但與可變剪接形式sDVLR1/CLEC5A(aa 43-65缺失)序列編號：73則否。相對的，JEV僅與sDVLR1/CLEC5A具交互作用，但與全長DVLR1/CLEC5A則否。如圖26b所示，其顯示DV可在人類巨噬細胞中誘導DAP12磷酸化作用(在2 h p.i.)。以抗-DAP12 mAb沈澱經DV感染之巨噬細胞中的DAP12，在SDS-PAGE分離後將其轉漬至硝基纖維素膜上，接著再與對抗磷醯酪胺酸及DAP12之抗體分別進行培育。JEV誘導之DAP12磷酸化作用(m.o.i.=5)由pLL3.7/DVLR1/CLEC5A抑制。如圖26c所示，其顯示人類巨噬細胞反應JEV感染之TNF-α分泌的動力學(左)。JEV誘導之TNF-α分泌由pLL3.7/DVLR1/CLEC5A mAb抑制(右)。數據係以三次獨立試驗之平均值±S.d.表示。Similar to DV, JEV has a similar pattern of viral infection response, and its scent is the same or similar in all flaviviruses. As shown in FIG 26a, respectively an ELISA assay DVLR1 / CLEC5A (1 μg) interacting with JEV and DV (5x10 ⁶ PFU) of. DV and human DVLR1/CLEC5A (length 187 amino acid) sequence number: 72 interactions, but with alternative splicing form sDVLR1/CLEC5A (aa 43-65 deletion) sequence number: 73 no. In contrast, JEV only interacts with sDVLR1/CLEC5A, but not with full-length DVLR1/CLEC5A. As shown in Figure 26b, it was shown that DV can induce DAP12 phosphorylation (at 2 h pi) in human macrophages. DAP12 in DV-infected macrophages was precipitated with anti-DAP12 mAb, and then stained onto nitrocellulose membrane by SDS-PAGE separation, followed by antibodies against phosphonium tyrosine and DAP12, respectively. Cultivate. JEV-induced DAP12 phosphorylation (moi=5) was inhibited by pLL3.7/DVLR1/CLEC5A. As shown in Figure 26c, it shows the kinetics of human macrophage response to TNF-[alpha] secretion by JEV infection (left). JEV-induced TNF-α secretion was inhibited by the pLL3.7/DVLR1/CLEC5A mAb (right). Data are expressed as the mean ± Sd of three independent experiments.

實施例27：mAb 3E12A2之可變重及輕鏈序列Example 27: Variable heavy and light chain sequences of mAb 3E12A2

mAb 3E12A2之可變重鏈序列示於下文(序列編號：60)： mAb 3E12A2之可變輕鏈序列示於下文(序列編號：61)： The variable heavy chain sequence of mAb 3E12A2 is shown below (SEQ ID NO: 60): The variable light chain sequence of mAb 3E12A2 is shown below (SEQ ID NO: 61):

實施例28：mAb 3E12G9之可變重及輕鏈序列Example 28: Variable heavy and light chain sequences of mAb 3E12G9

mAb 3E12G9之可變重鏈序列示於下文(序列編號：62)： mAb 3E12G9之可變輕鏈序列示於下文(序列編號：63)： The variable heavy chain sequence of mAb 3E12G9 is shown below (SEQ ID NO: 62): The variable light chain sequence of mAb 3E12G9 is shown below (SEQ ID NO: 63):

實施例29：mAb 8H8F5之可變重及輕鏈序列Example 29: Variable heavy and light chain sequences of mAb 8H8F5

mAb 8H8F5之可變重鏈序列示於下文(序列編號：64)： mAb 8H8F5之可變輕鏈序列示於下文(序列編號：65)： The variable heavy chain sequence of mAb 8H8F5 is shown below (SEQ ID NO: 64): The variable light chain sequence of mAb 8H8F5 is shown below (SEQ ID NO: 65):

實施例30：mAb 8H8F5、3E12A2、及3E12G9之可變重及輕鏈序列排比之比較 mAb 8H8F5、3E12A2、及3E12G9之可變重鏈排比之比較示於下文(分別為序列編號：66、67、及68)： Example 30: Comparison of variable heavy and light chain sequence ratios of mAbs 8H8F5, 3E12A2, and 3E12G9 Comparison of variable heavy chain ratios of mAbs 8H8F5, 3E12A2, and 3E12G9 are shown below (SEQ ID NO: 66, 67, respectively) And 68):

mAb 8H8F5、3E12A2、及3E12G9之可變輕鏈排比之比較示於下文(分別為序列編號：69、70、及71)： The comparison of the variable light chain ratios of mAbs 8H8F5, 3E12A2, and 3E12G9 is shown below (SEQ ID NO: 69, 70, and 71, respectively):

DVLR1/CLEC5A可與登革熱病毒直接交互作用，並因而造成DAP12磷酸化。DVLR1/CLEC5A-DV交互作用之封阻可壓抑促發炎性細胞激素之分泌，且不影響干擾素-α之釋出。再者，抗-DVLR1/CLEC5A單株抗體可在STAT1-缺陷小鼠體內抑制DV誘導之血漿滲漏以及皮下及生命器官出血，並可撿賞~50%之DV感染發生。該等結果建議，DV啟動之巨噬細胞的細胞因子釋出涉及DVLR1/CLEC5A及TLR7途徑兩者，而DVLR1/CLEC5A-DV交互作用之封阻可使感染減活且不會阻止病毒之清除。然而，TLR7(或MyD88)受體之封阻會抑制促發炎性細胞激素以及病毒清除細胞激素兩者之分泌，其最後會阻止病毒清除以及感染。因此，登革熱病毒以及諸如日本腦脊髓炎病毒之其他黃病毒的有效治療需要減活對於DVLR1/CLEC5A之病毒結合，但不應減活對於TLR7或MyD88受體之病毒結合。因此，以抗-DVLR1/CLEC5A抗體封阻登革熱病毒之結合可提供一種療法對抗DHF/DSS病患體內之嚴重登革熱進展。DVLR1/CLEC5A interacts directly with dengue virus and thus causes DAP12 phosphorylation. The blockade of DVLR1/CLEC5A-DV interaction suppresses the secretion of inflammatory cytokines and does not affect the release of interferon-α. Furthermore, anti-DVLR1/CLEC5A monoclonal antibodies can inhibit DV-induced plasma leakage and subcutaneous and vital organ bleeding in STAT1-deficient mice, and can reward ~50% of DV infections. These results suggest that cytokine release from DV-primed macrophages involves both the DVLR1/CLEC5A and TLR7 pathways, while the blockade of DVLR1/CLEC5A-DV interactions can inactivate infection and prevent viral clearance. However, blockade of the TLR7 (or MyD88) receptor inhibits the secretion of both pro-inflammatory cytokines and viral clearance cytokines, which ultimately prevents viral clearance and infection. Thus, effective treatment of dengue viruses and other flaviviruses such as Japanese encephalomyelitis virus requires attenuating viral binding to DVLR1/CLEC5A, but should not attenuate viral binding to TLR7 or MyD88 receptors. Thus, blocking the binding of dengue virus with an anti-DVLR1/CLEC5A antibody provides a therapy against severe dengue progression in DHF/DSS patients.

上述書面說明被認為足以使熟習本技術人士實施本發明。既然寄存之具體例僅係意欲例示說明本發明之某些態樣以及在功能上對等的任何構築體均在本發明之範圍內，本發明在範圍上不受寄存之雜交瘤所限。物質之寄存非表示承認本文之書面說明不足以實施本發明之任何態樣(包括其最佳方式)，亦不表示將申請專利範圍限於其所代表之特定例示說明例。事實上，本發明之各種修飾，除了本文所例示及說明者外，其餘亦為熟習本技術者所顯而易知以及落在附屬申請專利範圍之範圍內。The above written description is considered to be sufficient for a person skilled in the art to practice the invention. Since the specific examples of storage are merely intended to illustrate certain aspects of the invention and any constructs that are functionally equivalent, are within the scope of the invention, and the invention is not limited by the number of hybridomas that are deposited. The stipulations of the present invention are not to be construed as limiting the scope of the invention to the specific exemplary embodiments. In addition, the various modifications of the present invention are intended to be apparent to those skilled in the art and are within the scope of the appended claims.

本專利或專利申請檔案含有至少一張以彩色製成之圖式。本專利或專利申請公開案(含彩色圖式)之影本將依請求及繳付必要費用後由智財局提供。本說明書揭露之上述特徵及目的將可在參照下列之敘述並結合附呈之圖式閱讀時更為明瞭，該等圖式中之類似參照數目代表類似之元件，且其中：This patent or patent application file contains at least one drawing made in color. A copy of this patent or patent application publication (including color drawings) will be provided by the Intellectual Property Office upon request and payment of the necessary fees. The above-mentioned features and objects of the present invention will be more apparent from the following description of the appended claims.

圖1A顯示藉由RT-PCR擴增，然後在0.8%瓊脂糖上分成各部分及藉由溴化乙錠染色而顯現之先天性免疫受體之DNA片段。圖1B為經表現之重組受體.Fc融合蛋白質在12%SDS-PAGE凝膠上之電泳圖譜。Figure 1A shows a DNA fragment of an innate immune receptor visualized by RT-PCR amplification followed by fractionation on 0.8% agarose and staining with ethidium bromide. Figure 1B is an electropherogram of the expressed recombinant receptor.Fc fusion protein on a 12% SDS-PAGE gel.

圖2A顯示固定於膜上之生物素化GLPSF3與綴合有辣根過氧化酶(HRP)之鏈黴抗生物素接觸後之點漬圖。圖2B顯示固定於膜上之生物素化GLPS F3與樹狀細胞凝集素-1(Dectin-1).Fc融合蛋白質接觸，繼而與綴合有HRP之山羊抗IgG1抗體一起培育後之點漬圖。圖2C顯示圖2B之吸漬圖之點密度分析。圖2D顯示競爭者β-葡聚糖對於樹狀細胞凝集素-1.Fc融合蛋白質與固定於膜之GLPS F3之結合之點密度之影響。圖2E顯示固定之GLPS F3與樹狀細胞凝集素-1：Fc融合蛋白質接觸，繼而於存在各種量之競爭者多醣體(β-葡聚糖、D-葡萄糖及D-半乳糖)下與綴合有HRP之山羊抗IgG1抗體一起培育後之點漬圖。Figure 2A shows a spotting pattern of biotinylated GLSSF3 immobilized on a membrane after contact with streptavidin conjugated with horseradish peroxidase (HRP). Figure 2B shows the staining of biotinylated GLPS F3 immobilized on a membrane in contact with a dendritic cell lectin-1 (Dectin-1).Fc fusion protein, followed by incubation with a goat anti-IgG1 antibody conjugated with HRP. . Figure 2C shows the dot density analysis of the blotting diagram of Figure 2B. Figure 2D shows the effect of competitor β-glucan on the dot density of the binding of dendritic lectin-1.Fc fusion protein to GLPS F3 immobilized on the membrane. Figure 2E shows that the immobilized GLPS F3 is contacted with the dendritic cell lectin-1:Fc fusion protein, followed by the presence of various amounts of competitor polysaccharides (β-glucan, D-glucose, and D-galactose). A spotted map of a goat anti-IgG1 antibody combined with HRP.

圖3顯示固定於膜之GLPS F3及GLPS F3C與27種不同的融合蛋白質接觸後之點漬之半定量分析，其中該等融合蛋白質各包含所列之先天性免疫受體之細胞外區域且該細胞外區域與IgG1 Fc偶合。Figure 3 shows a semi-quantitative analysis of spotted stains of GLPS F3 and GLPS F3C immobilized on a membrane after contact with 27 different fusion proteins, each of which contains an extracellular region of the listed innate immune receptor and The extracellular region is coupled to IgG1 Fc.

圖4A顯示用圖3所列之27種融合蛋白質偵測固定於膜之GLPS F3及GLPS F3C之點漬圖。圖4B顯示EDTA對於樹狀細胞凝集素-1(Dectin-1).Fc、DC-SIGNR.Fc、KCR.Fc及TLT-2.Fc與固定於膜之GLPS F3之結合之影響。圖4C顯示用樹狀細胞凝集素-1(Dectin-1).Fc、DC-SIGNR.Fc、KCR.Fc及TLT-2.Fc融合蛋白質偵測固定於膜之β-葡聚糖之點漬圖。Figure 4A shows the spotting of GLPS F3 and GLPS F3C immobilized on the membrane using the 27 fusion proteins listed in Figure 3. Figure 4B shows the effect of EDTA on the binding of dendritic cell lectin-1 (Dectin-1).Fc, DC-SIGNR.Fc, KCR.Fc and TLT-2.Fc to membrane-bound GLPS F3. Figure 4C shows the detection of β-glucan immobilized on the membrane with dendritic cell lectin-1 (Dectin-1).Fc, DC-SIGNR.Fc, KCR.Fc and TLT-2.Fc fusion proteins. Figure.

圖5A顯示用樹狀細胞凝集素-1.Fc、DC-SIGNR.Fc、KCR.Fc及TLT-2.Fc融合蛋白質偵測多醣體樣品之點漬圖。圖5B顯示樣品編號之名稱及以半定量形式提供圖5A之點密度。Figure 5A shows a spotting pattern of a polysaccharide sample detected using dendritic cell lectin-1.Fc, DC-SIGNR.Fc, KCR.Fc and TLT-2.Fc fusion proteins. Figure 5B shows the name of the sample number and provides the dot density of Figure 5A in semi-quantitative form.

圖6A顯示包覆在微量滴定平皿上之生物素化GLPS-F3之量，其係使用過氧化酶-抗生物素蛋白綴合物檢定分析法測量及在OD 450 nm讀數，以檢測黃色反應產物。圖6B係以圖之方式描述各種受體.Fc融合蛋白質對固定於於微量平皿上之GLPS-F3之親和性。各受體.Fc融合蛋白質之絕對結合率記載在左側Y軸(以OD 450nm讀數表示)，以及右側Y軸記載與樹狀細胞凝集素-1.Fc之結合率相較下之相對結合率。Figure 6A shows the amount of biotinylated GLPS-F3 coated on a microtiter plate using a peroxidase-avidin conjugate assay and reading at OD 450 nm to detect yellow reaction products. . Figure 6B is a graphical representation of the affinity of various receptor.Fc fusion proteins for GLPS-F3 immobilized on microplates. The absolute binding rate of each receptor.Fc fusion protein is described on the left Y-axis (expressed as OD 450 nm reading), and the right Y-axis describes the relative binding rate compared to the binding rate of dendritic cell lectin-1.Fc.

圖7係以圖說明在與為多醣體之甘露聚糖及β-葡聚糖，以及與為單醣之D-甘露糖(Man)、D-葡萄糖(Glc)、N-乙醯基-葡萄糖胺(GlcNAc)、D-半乳糖(Gal)、N-乙醯基-半乳糖胺(GalNAc)、L-岩藻糖(Fuc)及唾液酸之競爭性檢定分析中，各種受體.Fc融合蛋白質與GLPS-F3之結合百分率。Figure 7 is a diagram illustrating the presence of mannan and β-glucan as polysaccharides, and D-mannose (Man), D-glucose (Glc), N-ethylidene-glucose as monosaccharides. Various receptors.Fc fusions in competitive assays for amines (GlcNAc), D-galactose (Gal), N-ethylmercapto-galactosamine (GalNAc), L-fucose (Fuc), and sialic acid The percentage of protein bound to GLPS-F3.

圖8A係以圖說明在與人類IgG陰性對照組相較下，受體.Fc融合蛋白質與登革熱病毒之結合率。圖8B顯示登革熱病毒與三種受體.Fc融合蛋白質及人類IgG陰性對照組之免疫複合物之西方轉漬圖，其中使用對抗登革熱病毒E蛋白質之抗體偵測。圖8C以圖顯示EDTA抑制登革熱病毒與DC-SIGN.Fc融合蛋白質之結合，但不會抑制登革熱病毒與DVLR1.Fc融合蛋白質之結合。圖8D顯示DVLR1.Fc融合蛋白質與用PNGaseF、二硫蘇糖醇(DTT)、熱或UV照射處理之登革熱病毒之結合率以及與未經處理之登革熱病毒(non)之結合率。Figure 8A is a graphical representation of the binding rate of a receptor.Fc fusion protein to a dengue virus in comparison to a human IgG negative control. Figure 8B shows a Western blot of the immune complex of dengue virus with three receptor. Fc fusion proteins and a human IgG negative control using antibody detection against dengue virus E protein. Figure 8C is a graph showing that EDTA inhibits the binding of dengue virus to the DC-SIGN.Fc fusion protein, but does not inhibit the binding of dengue virus to the DVLR1.Fc fusion protein. Figure 8D shows the binding rate of DVLR1.Fc fusion protein to dengue virus treated with PNGaseF, dithiothreitol (DTT), heat or UV irradiation, and binding rate to untreated dengue virus (non).

圖9A顯示DVLR1在各種免疫細胞類型中之表現，其藉由使用抗-DVLR1抗體之流動式細胞計量術測量。DVLR1之表現被示出，其中DVLR1之圖形輪廓(如虛線所示)與抗體同種型(isotype)對照組(陰影區)不相符。圖9B顯示DC-SIGN在各種免疫細胞類型中之表現，其藉由使用抗-DC-SIGN抗體之流式細胞計量術測量。DC-SIGN之表現被示出，其中DC-SIGN曲線(虛線)與抗體同種型對照組(陰影區)不相符。Figure 9A shows the performance of DVLR1 in various immune cell types as measured by flow cytometry using anti-DVLR1 antibodies. The performance of DVLR1 is shown, where the graphical outline of DVLR1 (as indicated by the dashed line) does not match the isotype control (shaded area) of the antibody. Figure 9B shows the performance of DC-SIGN in various immune cell types as measured by flow cytometry using anti-DC-SIGN antibodies. The performance of DC-SIGN is shown where the DC-SIGN curve (dashed line) does not match the antibody isotype control (shaded area).

圖10A顯示在與活登革熱病毒或經UV照射之登革熱病毒(UV-DV)接觸之CD14+巨噬細胞中NS3蛋白質表現之流式細胞計量分析(其中使用抗-NS3抗體)結果，並與匹配之抗體同種型對照組(陰影區)比較。圖10B圖示在以不同感染重複數(multiplicities of infection，MOI)之登革熱病毒感染之CD14+巨噬細胞中或以經UV照射之登革熱病毒感染之CD14+巨噬細胞中，細胞外登革熱病毒滴度之經時變化。圖10C係說明在以不同感染重複數(MOI)登革熱病毒感染之CD14+巨噬細胞中全部DAP12及磷醯化DAP12之免疫轉漬圖。圖10D係說明用活登革熱病毒或經UV照射之登革熱病毒以MOI=5感染後，於不同時間在該等經登革熱病毒感染之CD14+巨噬細胞中所有DAP12及磷醯化DAP12之免疫轉漬圖。Figure 10A shows the results of flow cytometric analysis of NS3 protein expression in CD14+ macrophages in contact with live dengue virus or UV-irradiated dengue virus (UV-DV), in which anti-NS3 antibodies were used, and matched Antibody isotype control (shaded area) comparison. Figure 10B illustrates extracellular dengue virus titer in CD14+ macrophages infected with dengue virus (HIV) with different multiplicities of infection (MOI) or in CD14+ macrophages infected with UV-irradiated dengue virus Change over time. Figure 10C is a graph showing the immunoblotting of all DAP12 and phosphonylated DAP12 in CD14+ macrophages infected with dengue virus at different infection repeat numbers (MOI). Figure 10D is a diagram showing the immunoblotting of all DAP12 and phosphonylated DAP12 in dengue virus-infected CD14+ macrophages at different times after infection with MOI=5 by live dengue virus or UV-irradiated dengue virus. .

圖11係例示說明用活登革熱病毒感染之前，藉由電穿孔導入pLL3.7載體(對照組)或DVLR1-shRNA之CD14+巨噬細胞中，所有DAP12及磷醯化DAP12之免疫轉漬圖。Figure 11 is a diagram showing the immunoblotting of all DAP12 and phosphonylated DAP12 in CD14+ macrophages introduced into pLL3.7 vector (control) or DVLR1-shRNA by electroporation prior to infection with live dengue virus.

圖12A顯示用活登革熱病毒或經UV照射之登革熱病毒以指定之MOI感染CD14+巨噬細胞後6小時，TNF-α之分泌。圖12B顯示用活登革熱病毒或經UV照射之登革熱病毒以指定之MOI感染CD14+巨噬細胞後12小時，TNF-α之分泌。圖12C顯示CD14+巨噬細胞感染後TNF-α分泌隨時程之測量值。Figure 12A shows secretion of TNF-[alpha] 6 hours after infection of CD14+ macrophages with live dengue virus or UV-irradiated dengue virus at the indicated MOI. Figure 12B shows secretion of TNF-[alpha] 12 hours after infection of CD14+ macrophages with live dengue virus or UV-irradiated dengue virus at the indicated MOI. Figure 12C shows measurements of TNF-[alpha] secretion over time after infection with CD14+ macrophages.

圖13A顯示在DC-SIGN-shRNA或DVLR1-shRNA、或者載體對照組(pWTSI及pLL3.7)轉染之CD14+巨噬細胞中，藉由西方轉漬測得之DC-SIGN及DVLR1之表現。圖13B顯示在用登革熱病毒染之前，藉由電穿孔導入DC-SIGN-shRNA、DVLR1-shRNA、或者pLL3.7載體對照組之CD14+巨噬細胞中，NS3表現之流動式細胞計量分析(使用抗-NS3抗體)。陰影區為NS3抗體之同種型對照組。圖13C係例示說明在用登革熱病毒感染之前(t=0)，藉由電穿孔導入DC-SIGN-shRNA、DVLR1-shRNA或載體對照組之CD14+巨噬細胞之上清液中病毒滴度之時程分析。Figure 13A shows the expression of DC-SIGN and DVLR1 as measured by Western blotting in DC-SIGN-shRNA or DVLR1-shRNA, or in vehicle control (pWTSI and pLL3.7) transfected CD14+ macrophages. Figure 13B shows flow cytometric analysis of NS3 expression by electroporation of DC-SIGN-shRNA, DVLR1-shRNA, or pLL3.7 vector control group in CD14+ macrophages prior to infection with dengue virus. -NS3 antibody). The shaded area is the isotype control group of the NS3 antibody. Figure 13C is a diagram showing the timing of virus titer in the supernatant of CD14+ macrophages introduced into DC-SIGN-shRNA, DVLR1-shRNA or vehicle control group by electroporation before infection with dengue virus (t=0) Process analysis.

圖14A顯示用登革熱病毒感染之前(t=0)，藉由電穿孔導入DC-SIGN-shRNA、DVLR1-shRNA或載體對照組之CD14+巨噬細胞中各種細胞激素之之分泌之時程分析。圖14B顯示於相同條件下細胞激素IFN-α之時程分析。Figure 14A shows time course analysis of secretion of various cytokines in CD14+ macrophages introduced into DC-SIGN-shRNA, DVLR1-shRNA or vehicle control by electroporation prior to infection with dengue virus (t=0). Figure 14B shows the time course analysis of the cytokine IFN-α under the same conditions.

圖15顯示由感染有登革熱病毒且用對抗DVLR1之指定單株抗體以指定濃度治療之CD14+巨噬細胞分泌入培養上清液中之TNF-α之ELISA測量值。Figure 15 shows ELISA measurements of TNF-[alpha] secreted into culture supernatant by CD14+ macrophages infected with dengue virus and treated with the indicated monoclonal antibodies against DVLR1 at the indicated concentrations.

圖16a圖式說明各種受體.Fc融合蛋白對於登革熱病毒之結合。圖16b顯示登革熱病毒與三種受體.Fc融合蛋白及一種人類IgG陰性控制組之免疫復合物的西方轉漬，以對抗登革熱病毒E蛋白之抗體探測。圖16c圖示由EDTA可抑制登革熱病毒對DC-SIGN.Fc融合蛋白之結合，但不會抑制對於DLVR1/CLEC5A融合蛋白之結合。圖16d顯示藉由添加DC-SIGN.Fc及DLVR1/CLEC5A融合蛋白而產生登革熱病毒對人類293T細胞結合之增加。16e圖示各種糖之添加可抑制登革熱病毒對DC-SIGN.Fc融合蛋白之結合。16f圖示PNGaseF對於登革熱病毒對DLVR1/CLEC5A融合蛋白之結合的效應。Figure 16a is a graphical representation of the binding of various receptor.Fc fusion proteins to dengue virus. Figure 16b shows Western blotting of the immune complex of dengue virus with three receptor.Fc fusion proteins and a human IgG negative control group to counteract antibody detection of dengue virus E protein. Figure 16c illustrates that EDTA inhibits the binding of dengue virus to the DC-SIGN.Fc fusion protein, but does not inhibit binding to the DLVR1/CLEC5A fusion protein. Figure 16d shows the increase in binding of dengue virus to human 293T cells by the addition of DC-SIGN.Fc and DLVR1/CLEC5A fusion proteins. 16e shows that the addition of various sugars inhibits the binding of dengue virus to the DC-SIGN.Fc fusion protein. 16f shows the effect of PNGaseF on the binding of dengue virus to the DLVR1/CLEC5A fusion protein.

圖17a說明DC-SIGN在人類PBMCs中之表現模式。圖17b說明DVLR1/CLEC5A在人類PBMCs中之表現模式。Figure 17a illustrates the pattern of DC-SIGN expression in human PBMCs. Figure 17b illustrates the pattern of DVLR1/CLEC5A expression in human PBMCs.

圖18a顯示免疫轉漬，其說明使用針對磷醯酪胺酸及DAP12之抗體，測定人類巨噬細胞中之登革熱病毒誘導之DAP12磷醯化(2 h p.i.)。圖18b顯示免疫轉漬，其說明由登革熱病毒及經UV-失活之登革熱病毒誘導之DAP12磷醯化的動力學。圖18c顯示免疫轉漬，其說明shRNAs使DVLR1/CLEC5A及DC-SIGN.Fc融合蛋白之表現下降(knock down)以及抑制登革熱病毒(m.o.i.=5)-媒介之DAP12磷醯化的能力。圖18d顯示shRNAs對於巨噬細胞中之登革熱病毒進入及複製的效應。圖18e顯示抗-DVLR1/CLEC5A mAb、抗-DC-SIGN mAb、及小鼠IgG對於非結構蛋白質NS3表現的效應。圖18f圖示說明shRNAs對於經感染巨噬細胞之登革熱病毒滴度的效應之時程檢定分析。Figure 18a shows immunoblots, which demonstrate dengue virus-induced DAP12 phosphorylation (2 h p.i.) in human macrophages using antibodies against phosphonium tyrosine and DAP12. Figure 18b shows immunoblotting, which illustrates the kinetics of DAP12 phosphonium induced by dengue virus and UV-inactivated dengue virus. Figure 18c shows immunoblots demonstrating the ability of shRNAs to knock down the expression of the DVLR1/CLEC5A and DC-SIGN.Fc fusion proteins and inhibit the dendriticization of dengue virus (m.o.i.=5)-mediated DAP12. Figure 18d shows the effect of shRNAs on dengue virus entry and replication in macrophages. Figure 18e shows the effect of anti-DVLR1/CLEC5A mAb, anti-DC-SIGN mAb, and mouse IgG on the expression of non-structural protein NS3. Figure 18f graphically illustrates a time course assay for the effect of shRNAs on dengue virus titers of infected macrophages.

圖19a圖示說明巨噬細胞之登革熱病毒及經UV失活之登革熱病毒誘導性TNF-α分泌的劑量-依存性。圖19b說明登革熱病毒感染(m.o.i.=5)後之TNF-α表現的動力學。圖19圖示說明DVLR1/CLEC5A及DC-SIGN shRNA對於TNF-α、IL-6、MIP1-α、IL-8、IP-10及INF-α自經登革熱病毒感染(m.o.i.=5)之巨噬細胞分泌的效應。在圖19d圖示說明以特異性shRNA對於登革熱病毒誘導性TNF-α及INF-α分泌之各種分泌途徑所進行之下降(knock down)實驗效應。圖19e圖示說明桔抗性抗-DVLR1/CLEC5A mAbs可抑制反應登革熱病毒血清型1-4之TNF-α分泌。使用M.R.mAb(抗-甘露糖受體mAb，mIgG1)及小鼠IgM(mIgM)作為陰性控制組。Figure 19a graphically illustrates the dose-dependency of macrophage dengue virus and UV-inactivated dengue virus-induced TNF-[alpha] secretion. Figure 19b illustrates the kinetics of TNF-[alpha] expression following dengue virus infection (m.o.i.=5). Figure 19 is a graphical representation of DVLR1/CLEC5A and DC-SIGN shRNA for TNF-α, IL-6, MIP1-α, IL-8, IP-10 and INF-α from dengue virus infection (moi=5) The effect of cell secretion. Figure 19d graphically illustrates the knockdown experimental effect of specific shRNAs on various secretory pathways of dengue virus-induced TNF-[alpha] and INF-[alpha] secretion. Figure 19e illustrates that citrus-resistant anti-DVLR1/CLEC5A mAbs inhibit TNF-α secretion in dengue virus serotypes 1-4. M.R. mAb (anti-mannose receptor mAb, mIgG1) and mouse IgM (mIgM) were used as negative control groups.

圖20a說明經登革熱病毒、登革熱病毒/抗-E、及登革熱病毒/抗-prM免疫複合物感染之巨噬細胞的NS3表現。圖20b說明經DV2感染之巨噬細胞的TNF-α及INF-α分泌量。圖20c說明經抗-prM/登革熱病毒及抗-E/登革熱病毒免疫複合物感染之巨噬細胞的TNF-α及INF-α分泌量。Figure 20a illustrates NS3 expression of macrophages infected with dengue virus, dengue virus/anti-E, and dengue virus/anti-prM immune complexes. Figure 20b illustrates the amount of TNF-[alpha] and INF-[alpha] secreted by DV2-infected macrophages. Figure 20c illustrates the amount of TNF-[alpha] and INF-[alpha] secreted by macrophages infected with anti-prM/dengue virus and anti-E/dengue virus immune complexes.

圖21a圖示說明HMEC-1單層之通透性以及上清液中之TNF-α含量的時程分析。圖21b圖示說明TNFR2.Fc及抗-DVLR1/CLEC5A對於內皮細胞單層通透化的抑制作用。Figure 21a graphically illustrates the time course analysis of the permeability of the HMEC-1 monolayer and the TNF-α content in the supernatant. Figure 21b graphically illustrates the inhibition of endothelial cell monolayer permeabilization by TNFR2.Fc and anti-DVLR1/CLEC5A.

圖22a圖示說明登革熱病毒與人類及小鼠DVLR1/CLEC5A.Fc融合蛋白的結合親和力。圖22b顯示mDVLR1/CLEC5A在小鼠脾細胞中之表現。圖22c顯示mDVLR1/CLEC5A在小鼠骨髓(BM)-衍生性巨噬細胞及小鼠似巨噬細胞Raw264.7細胞系中之表現。Figure 22a illustrates the binding affinity of dengue virus to human and mouse DVLR1/CLEC5A.Fc fusion proteins. Figure 22b shows the performance of mDVLR1/CLEC5A in mouse spleen cells. Figure 22c shows the expression of mDVLR1/CLEC5A in mouse bone marrow (BM)-derived macrophages and mouse-like macrophage Raw264.7 cell lines.

圖23a圖示說明由登革熱病毒與小鼠巨噬細胞細胞系Raw264.7及穩定表現人類DC-SIGN之Raw264.7細胞之結合所產生之TNF-α釋出比較。圖23b圖示說明在mAb之存在下使穩定表現人類DC-SIGN之Raw264.7細胞與DV2共同進行培育所產生之TNF-α分泌。圖23c圖示說明抗-DVLR1/CLEC5A mAbs(3D2H6及10D7H3)可以劑量-依存性之方式抑制DV2-誘導之TNF-α釋出。Figure 23a is a graphical representation of the release of TNF-[alpha] produced by the binding of dengue virus to the mouse macrophage cell line Raw264.7 and the stable expression of human DC-SIGN Raw264.7 cells. Figure 23b is a graphical representation of TNF-[alpha] secretion produced by culturing Raw264.7 cells stably expressing human DC-SIGN in combination with DV2 in the presence of mAb. Figure 23c is a graphical representation of that anti-DVLR1/CLEC5A mAbs (3D2H6 and 10D7H3) inhibit DV2-induced TNF-α release in a dose-dependent manner.

圖24說明經DV2/PL046或DV2/NGC-N系挑戰之STAT1 ^-/- 小鼠的Kaplan-Meier存活曲線。Figure 24 illustrates Kaplan-Meier survival curves of STAT1 ^-/- mice challenged with DV2/PL046 or DV2/NGC-N lines.

圖25a說明對抗小鼠DVLR1/CLEC5A培育之mAb 3D2H6及10D7H3對於經登革熱病毒挑戰之STAT1 ^-/- 小鼠的皮下及腸道出血之效應。圖25b說明對抗DVLR1/CLEC5A之mAb(3D2H6及10D7H3)對於經登革熱病毒挑戰之STAT1 ^{-/ -} 小鼠的血漿滲漏進入生命器官之效應。圖25c圖示說明藉著自器官萃取Evans藍顯示之生命器官血管通透性。圖25d說明於有及無抗-DVLR1/CLEC5A mAbs或TNFR2.Fc存在下，經登革熱病毒挑戰之STAT1 ^-/- 小鼠之TNF-α及IP-10之血清含量以及病毒滴度。圖25e圖示說明在桔抗性抗-小鼠DVLR1/CLEC5A mAbs或TNFR2.Fc存在下，經DV2挑戰之STAT1缺陷小鼠的存活。Figure 25a illustrates the effect of mAb 3D2H6 and 10D7H3 against mouse DVLR1/CLEC5A on subcutaneous and intestinal bleeding in dengue virus challenged STAT1 ^-/- mice. Figure 25b illustrates the effect of mAb (3D2H6 and 10D7H3) against DVLR1/CLEC5A on plasma leakage of dengue virus challenged STAT1 ^{-/ -} mice into living organs. Figure 25c illustrates vascular permeability of vital organs as indicated by Evans blue extraction from the organ. Figure 25d illustrates serum levels and viral titers of TNF-[alpha] and IP-10 in dengue virus challenged STAT1 ^-/- mice in the presence and absence of anti-DVLR1/CLEC5A mAbs or TNFR2.Fc. Figure 25e is a graphical representation of the survival of DV2-challenged STAT1-deficient mice in the presence of citrus-resistant anti-mouse DVLR1/CLEC5A mAbs or TNFR2.Fc.

圖26說明DVLR1/CLEC5A涉及JEV媒介之DAP12磷酸化作用及自人類巨噬細胞之TNF-α分泌。在圖26a中，分別以ELISA測定DVLR1/CLEC5A(1 μg)與JEV及登革熱病毒(DV)(5x10⁶ PFU)之交互作用。DV與人類DVLR1/CLEC5A(長度188胺基酸)具交互作用，但與可變剪接形式sDVLR1/CLEC5A(aa 43-65缺失)則否。相對的，JEV僅與sDVLR1/CLEC5A具交互作用，但與全長DVLR1/CLEC5A則否。在圖26b中，登革熱病毒可在人類巨噬細胞中誘導DAP12磷酸化作用(在2 h p.i.)。以抗-DAP12 mAb沈澱經DV感染之巨噬細胞中的DAP12，在SDS-PAGE分離後將其轉漬至硝基纖維素膜上，接著再與對抗磷醯酪胺酸及DAP12之抗體分別進行培育。JEV誘導之DAP12磷酸化作用(m.o.i.=5)由pLL3.7/DVLR1/CLEC5A抑制。在圖26c中，其顯示人類巨噬細胞反應JEV感染之TNF-α分泌的動力學(左)。JEV誘導之TNF-α分泌由pLL3.7/DVLR1/CLEC5A mAb抑制(右)。數據係以三次獨立試驗之平均值±s.d.表示。Figure 26 illustrates that DVLR1/CLEC5A is involved in DAP12 phosphorylation of JEV mediators and TNF-alpha secretion from human macrophages. In FIG. 26a, respectively an ELISA assay DVLR1 / CLEC5A (1 μg) and with dengue virus (DV) (5x10 ⁶ PFU) interaction of JEV. DV interacts with human DVLR1/CLEC5A (length 188 amino acid), but with the alternative splicing form sDVLR1/CLEC5A (aa 43-65 deletion). In contrast, JEV only interacts with sDVLR1/CLEC5A, but not with full-length DVLR1/CLEC5A. In Figure 26b, dengue virus induces DAP12 phosphorylation (at 2 h pi) in human macrophages. DAP12 in DV-infected macrophages was precipitated with anti-DAP12 mAb, and then stained onto nitrocellulose membrane by SDS-PAGE separation, followed by antibodies against phosphonium tyrosine and DAP12, respectively. Cultivate. JEV-induced DAP12 phosphorylation (moi=5) was inhibited by pLL3.7/DVLR1/CLEC5A. In Figure 26c, it shows the kinetics of TNF-[alpha] secretion by human macrophage in response to JEV infection (left). JEV-induced TNF-α secretion was inhibited by the pLL3.7/DVLR1/CLEC5A mAb (right). Data are expressed as the mean ± sd of three independent experiments.

<110> 中央研究院<110> Academia Sinica

<120> 用於偵測目標反應與治療黃病毒感染症狀之化合物及方法<120> Compounds and methods for detecting target response and treating symptoms of flavivirus infection

<130> ACA0029TW<130> ACA0029TW

<150> US 11/469,270<150> US 11/469,270

<151> 2006-08-31<151> 2006-08-31

<160> 73<160> 73

<170> PatentIn version 3.5<170> PatentIn version 3.5

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Claims (7)

一種經純化的抗登革熱病毒凝集素受體1/C-型凝集素5A(DVLR1/CLEC5A)單株抗體，其中該單株抗體包括選自由以下所組成之群組之序列組合：(i)序列編號：66之可變重鏈序列及序列編號：69之可變輕鏈序列之組合；(ii)序列編號：67之可變重鏈序列及序列編號：70之可變輕鏈序列之組合；及(iii)序列編號：68之可變重鏈序列及序列編號：71之可變輕鏈序列之組合。 A purified monoclonal antibody against dengue virus lectin receptor 1/C-type lectin 5A (DVLR1/CLEC5A), wherein the monoclonal antibody comprises a sequence combination selected from the group consisting of: (i) a sequence No.: 66 variable heavy chain sequence and SEQ ID NO: 69 combination of variable light chain sequences; (ii) SEQ ID NO: 67 variable heavy chain sequence and SEQ ID NO: 70 combination of variable light chain sequences; And (iii) a combination of a variable heavy chain sequence of SEQ ID NO: 68 and a sequence of variable light chain of SEQ ID NO: 71. 如申請專利範圍第1項之單株抗體，其中該單株抗體包括(i)序列編號：66之可變重鏈序列及序列編號：69之可變輕鏈序列之組合。 The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises (i) a variable heavy chain sequence of SEQ ID NO: 66 and a combination of variable light chain sequences of SEQ ID NO: 69. 如申請專利範圍第1項之單株抗體，其中該單株抗體包括(ii)序列編號：67之可變重鏈序列及序列編號：70之可變輕鏈序列之組合。 The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises (ii) a variable heavy chain sequence of SEQ ID NO: 67 and a combination of variable light chain sequences of SEQ ID NO: 70. 如申請專利範圍第1項之單株抗體，其中該單株抗體包括(iii)序列編號：68之可變重鏈序列及序列編號：71之可變輕鏈序列之組合。 The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises (iii) a variable heavy chain sequence of SEQ ID NO: 68 and a combination of variable light chain sequences of SEQ ID NO: 71. 如申請專利範圍第1至4項中任一項之單株抗體，其中該單株抗體為人類化抗體。 The monoclonal antibody of any one of claims 1 to 4, wherein the monoclonal antibody is a humanized antibody. 如申請專利範圍第5項之單株抗體，其中該單株抗體可用於抑制受登革熱病毒病毒感染之巨噬細胞之促發炎性細胞激素的分泌。 A monoclonal antibody according to claim 5, wherein the monoclonal antibody is useful for inhibiting secretion of an inflammatory cytokine by a macrophage infected with a dengue virus. 如申請專利範圍第6項之單株抗體，其中該促發炎性細胞激素包含TNF-α。The monoclonal antibody of claim 6, wherein the pro-inflammatory cytokine comprises TNF-α.